Microbial compositions and methods for ruminant health and performance

ABSTRACT

The disclosure relates to isolated microorganisms—including novel strains of the microorganisms—microbial ensembles, and compositions comprising the same. Furthermore, the disclosure teaches methods of utilizing the described microorganisms, microbial compositions, and compositions comprising the same, in methods for modulating the agricultural production of ruminants. In particular aspects, the disclosure provides methods of increasing feed efficiency, and methods of decreasing acidosis.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a Continuation of U.S. application Ser. No.15/965,661, filed Apr. 27, 2018, which claims the benefit of priority toU.S. Provisional Application No. 62/491,845, filed Apr. 28, 2017; andU.S. Provisional Application No. 62/578,188, filed Oct. 27, 2017; eachof which is herein incorporated by reference in its entirety.

FIELD

The present disclosure relates to isolated and biologically puremicroorganisms that have applications, inter alia, in the farming ofbeef cattle. The disclosed microorganisms can be utilized in theirisolated and biologically pure states, as well as being formulated intocompositions. Furthermore, the disclosure provides a microbial ensemble,containing at least two members of the disclosed microorganisms, as wellas methods of utilizing said microbial ensemble. Furthermore, thedisclosure provides for methods of modulating the rumen microbiome.

STATEMENT REGARDING SEQUENCE LISTING

The sequence listing associated with this application is provided intext format in lieu of a paper copy, and is hereby incorporated byreference into the specification. The name of the text file containingthe sequence listing is ASBI_005_02US_ST25.txt. The text file is ˜4,544kb, was created on Apr. 27, 2018, and is being submitted electronicallyvia EFS-Web.

BACKGROUND

The global population is predicted to increase to over 9 billion peopleby the year 2050 with a concurrent reduction in the quantity of land,water, and other natural resources available per capita. Projectionsindicate that the average domestic income will also increase, with theprojected rise in the GDP of China and India. The desire for a dietricher in animal-source proteins rises in tandem with increasing income,thus the global livestock sector will be charged with the challenge ofproducing more animal products using fewer resources. The Food andAgriculture Organization of the United Nations predict that 70% morefood will have to be produced, yet the area of arable land availablewill decrease. It is clear that the food output per unit of resourceinput will have to increase considerably in order to support the rise inpopulation.

Over recent decades the farm industry has seen fast growth in the meatsector. As more of the world's population ascends into the middle classdemand for protein—including beef—is expected to remain robust for yearsto come. Worldwide beef production tops 59 million tons per annum.

Beef and products thereof are predominantly utilized in the preparationof foodstuffs in many different forms. There have been many strategiesto improve beef production through nutritional modulations, hormonetreatments, changes in animal management, and selective breeding;however, the need for more efficient production of edible beeffoodstuffs per animal is required. Current animal feeding and handlingpractices, for example, often induce microbial dysbiosis in the rumenthat ultimately leads to incidences of sub-acute acidosis or bloat,hindering the efficiency of production, increasing feed costs, and/orincreasing a reliance on chemistry based treatments, such asantibiotics.

Identifying compositions and methods for sustainably increasing beefproduction while balancing animal health and wellbeing have becomeimperative to satisfy the needs of everyday humans in an expandingpopulation. Increasing the worldwide production of beef by scaling upthe total number of beef cattle on farms would not only be economicallyinfeasible for many parts of the world, but would further result innegative environmental consequences as the beef sector's growth andtrends towards intensification and concentration have already given riseto a number of environmental concerns, led predominantly by theproduction of far more waste than can be managed by land disposal.

Population densities of beef cattle, particularly feedlot cattle, onlarge farms are often accompanied by an increased incidence of microbialpathogens that place the beef yield at risk, and further place theultimate consumer of the beef at risk in instances of zoonotic pathogensand/or the blooming of organisms in the rumen that lead to incidences ofsubacute acidosis (ruminal subacute acidosis) or bloat, which furtherhinders the productivity of feedlot operations. Considering thewidespread occurrence of many zoonotic pathogens, it is unlikely thatbeef can be completely protected from exposure. Research has focused oninvestigative means of increasing resistance to colonization in beefcattle exposed to these pathogens.

Thus, meeting global beef yield expectations, by simply scaling upcurrent high-input agricultural systems—utilized in most of thedeveloped world—is simply not feasible.

There is therefore an urgent need in the art for improved methods ofincreasing beef production, while also mitigating the colonization andspread of microbial pathogens and further increasing the desirableaspects of beef.

SUMMARY OF THE DISCLOSURE

In some aspects, the present disclosure provides isolated microbes,including novel strains of microbes, presented in Table 1 and/or Table2.

In other aspects, the present disclosure provides isolated wholemicrobial cultures of the microbes identified in Table 1 and Table 2.These cultures may comprise microbes at various concentrations.

In some aspects, the disclosure provides for utilizing one or moremicrobes selected from Table 1 and/or Table 2 to increase a phenotypictrait of interest in beef cattle.

In some embodiments, a microbial composition comprises at least twomicrobial strains selected from Table 1 and/or Table 2. In anotherembodiment, a microbial composition is provided, said compositioncomprising at least one microbial strain selected from Table 1 and/orTable 2. In a further embodiment, a microbial composition comprises atleast two microbial strains, wherein the at least two microbial strainscomprise a 16S rRNA sequence encoded by sequences selected from SEQ IDNOs:1-5993

In some embodiments, the disclosure is drawn to a ruminant supplementcapable of treating or preventing acidosis or bloat in a ruminant,comprising: a) a purified population of bacteria selected from any oneor more bacteria comprising a 16S nucleic acid sequence that is at leastabout 97% identical to any one of SEQ ID NO:1-5993: and b) a carriersuitable for ruminant administration; wherein the purified population ofbacteria of a) is present in the supplement in an amount effective totreat or prevent acidosis or bloat in a ruminant administered thesupplement, as compared to a ruminant not administered the supplement.

In some embodiments, the ruminant supplement capable of treating orpreventing acidosis or bloat in a ruminant, comprising: a) a purifiedpopulation of bacteria selected from: (i) Succinivibrio bacteria with a16S nucleic acid sequence that is at least about 97% identical to SEQ IDNO:75, (ii) Prevotella bacteria with a 16S nucleic acid sequence that isat least about 97% identical to SEQ ID NO:86, and/or (iii) Bacteroidesbacteria with a 16S nucleic acid sequence that is at least about 97%identical to SEQ ID NO:13; and b) a carrier suitable for ruminantadministration; wherein the purified population of bacteria of a) ispresent in the supplement in an amount effective to treat or preventacidosis or bloat in a ruminant administered the supplement, as comparedto a ruminant not administered the supplement.

On some embodiments, the at least one of the bacteria are selected from:(i) Succinivibrio bacteria with a 16S nucleic acid sequence that is atleast about 97% identical to SEQ ID NO:75, (ii)Prevotella bacteria witha 16S nucleic acid sequence that is at least about 97% identical to SEQID NO:86, and/or (iii) Bacteroides bacteria with a 16S nucleic acidsequence that is at least about 97% identical to SEQ ID NO:13

In one embodiment the purified population of bacteria is selected from:(i) Succinivibrio bacteria with a 16S nucleic acid sequence that is atleast about 99% identical to SEQ ID NO:75, (ii) Prevotella bacteria witha 16S nucleic acid sequence that is at least about 99% identical to SEQID NO:86, and/or (iii) Bacteroides bacteria with a 16S nucleic acidsequence that is at least about 99% identical to SEQ ID NO:13.

In one embodiment, purified population of bacteria is selected from: (i)Succinivibrio bacteria with a 16S nucleic acid sequence comprising SEQID NO:75, (ii) Prevotella bacteria with a 16S nucleic acid sequencecomprising SEQ ID NO:86, and/or (iii) Bacteroides bacteria with a 16Snucleic acid sequence comprising SEQ ID NO:13.

In one embodiment, the purified population of bacteria is selected from:(i) Succinivibrio bacteria deposited as B-67550. (ii) Prevotellabacteria deposited as B-67552, and/or (iii) Bacteroides bacteriadeposited as B-67555.

In one embodiment the purified population of bacteria comprises bacteriawith a 16S nucleic acid sequence that is at least about 97% identical toa nucleic acid sequence selected from the group consisting of SEQ IDNO:1-5993.

In one embodiment, the ruminant supplement further comprising a purifiedpopulation of bacteria that comprises bacteria with a 16S nucleic acidsequence that is at least about 99% identical to a nucleic acid sequenceselected from the group consisting of SEQ ID NO:1-5993.

In one embodiment, the ruminant supplement further comprising a purifiedpopulation of bacteria that comprises bacteria with a 16S nucleic acidsequence comprising a nucleic acid sequence selected from the groupconsisting of SEQ ID NO:1-5993.

In one embodiment the purified population of bacteria are encapsulatedin one or more of a polymer, carbohydrate, sugar, plastic, glass,polysaccharide, lipid, wax, oil, fatty acid, or glyceride.

In one embodiment, the encapsulated bacteria are vitrified. In oneembodiment, the encapsulated bacteria are further encapsulated in a wax.In one embodiment, the purified population of bacteria are in the formof spores. In one embodiment, the spores are spray dried. In oneembodiment, the ruminant supplement is formulated as a tablet, capsule,pill, feed additive, food ingredient, food additive, food preparation,food supplement, consumable solution, consumable spray additive,consumable solid, consumable gel, injection, bolus, or combinationsthereof.

In some embodiments, the disclosure is drawn to a method for treating orpreventing acidosis or bloat in a ruminant, comprising: administering toa ruminant an effective amount of a ruminant supplement comprising: a) apurified population of bacteria selected from: (i) Succinivibriobacteria with a 16S nucleic acid sequence that is at least about 97%identical to SEQ ID NO:75, (ii) Prevotella bacteria with a 16S nucleicacid sequence that is at least about 97% identical to SEQ ID NO:86,and/or (iii) Bacteroides bacteria with a 16S nucleic acid sequence thatis at least about 97% identical to SEQ ID NO:13; and b) a carriersuitable for ruminant administration; wherein the purified population ofbacteria of a) is present in the supplement in an amount effective totreat or prevent acidosis or bloat in a ruminant administered thesupplement, as compared to a ruminant not administered the supplement.

In some embodiments, at least one of the bacteria are selected from: (i)Succinivibrio bacteria with a 16S nucleic acid sequence that is at leastabout 97% identical to SEQ ID NO:75, (ii)Prevotella bacteria with a 16Snucleic acid sequence that is at least about 97% identical to SEQ IDNO:86, and/or (iii) Bacteroides bacteria with a 16S nucleic acidsequence that is at least about 97% identical to SEQ ID NO:13.

In some embodiments, the purified population of bacteria is selectedfrom: (i) Succinivibrio bacteria with a 16S nucleic acid sequence thatis at least about 99% identical to SEQ ID NO:75, (ii)Prevotella bacteriawith a 16S nucleic acid sequence that is at least about 99% identical toSEQ ID NO:86, and/or (iii) Bacteroides bacteria with a 16S nucleic acidsequence that is at least about 99% identical to SEQ ID NO:13.

In some embodiments, the purified population of bacteria is selectedfrom: (i) Succinivibrio bacteria with a 16S nucleic acid sequencecomprising SEQ ID NO:75, (ii) Prevotella bacteria with a 16S nucleicacid sequence comprising SEQ ID NO:86, and/or (iii) Bacteroides bacteriawith a 16S nucleic acid sequence comprising SEQ ID NO:13.

In some embodiments, the purified population of bacteria is selectedfrom: (i) Succinivibrio bacteria deposited as B-67550. (ii) Prevotellabacteria deposited as B-67552, and/or (iii) Bacteroides bacteriadeposited as B-67555.

In some embodiments, the ruminant supplement further comprises apurified population of bacteria that comprises bacteria with a 16Snucleic acid sequence that is at least about 97% identical to a nucleicacid sequence selected from the group consisting of SEQ ID NO:1-5993.

In some embodiments, the ruminant supplement further comprises apurified population of bacteria that comprises bacteria with a 16Snucleic acid sequence that is at least about 99% identical to a nucleicacid sequence selected from the group consisting of SEQ ID NO:1-5993.

In some embodiments, the ruminant supplement further comprises apurified population of bacteria that comprises bacteria with a 16Snucleic acid sequence comprising a nucleic acid sequence selected fromthe group consisting of SEQ ID NO:1-5993.

In some embodiments, the purified population of bacteria areencapsulated in one or more of a polymer, carbohydrate, sugar, sugaralcohol, surfactant, plastic, glass, polysaccharide, lipid, wax, oil,fatty acid, amino acid, or glyceride.

In one embodiment, the encapsulated bacteria are vitrified. In oneembodiment, the encapsulated bacteria are further encapsulated in a wax,fat, fatty acid, fatty alcohol, or glyceride. In one embodiment, thepurified population of bacteria are in the form of spores. In oneembodiment, the spores are spray dried. In one embodiment, thesupplement is formulated as a tablet, capsule, pill, feed additive, foodingredient, food additive, food preparation, food supplement, consumablesolution, consumable spray additive, consumable solid, consumable gel,injection, bolus, or combinations thereof.

In one embodiment, the ruminant is administered to a ruminant orally. Inone embodiment, the ruminant is a cow or a steer. In one embodiment, theruminant is fed a step-up diet. In one embodiment, the ruminant is fed afinishing diet.

The ruminant supplement of claim 36, wherein the purified population ofbacteria is selected from: (i) Succinivibrio bacteria with a 16S nucleicacid sequence that is at least about 99% identical to SEQ ID NO:75, (ii)Prevotella bacteria with a 16S nucleic acid sequence that is at leastabout 99% identical to SEQ ID NO:86, and/or (iii) Bacteroides bacteriawith a 16S nucleic acid sequence that is at least about 99% identical toSEQ ID NO:13.

In some embodiments, the purified population of bacteria is selectedfrom: (i) Succinivibrio bacteria with a 16S nucleic acid sequencecomprising SEQ ID NO:75, (ii) Prevotella bacteria with a 16S nucleicacid sequence comprising SEQ ID NO:86, and/or (iii) Bacteroides bacteriawith a 16S nucleic acid sequence comprising SEQ ID NO:13.

In some embodiments, the purified population of bacteria is selectedfrom (i) Succinivibrio bacteria deposited as B-67550, (ii) Prevotellabacteria deposited as B-67552, and/or (iii) Bacteroides bacteriadeposited as B-67555.

In some embodiments, the ruminant supplement further comprises apurified population of bacteria that comprises bacteria with a 16Snucleic acid sequence that is at least about 97% identical to a nucleicacid sequence selected from the group consisting of SEQ ID NO:1-5993.

In some embodiments, the ruminant supplement further comprises apurified population of bacteria that comprises bacteria with a 16Snucleic acid sequence that is at least about 99% identical to a nucleicacid sequence selected from the group consisting of SEQ ID NO:1-5993.

In some embodiments, the ruminant supplement further comprises apurified population of bacteria that comprises bacteria with a 16Snucleic acid sequence comprising a nucleic acid sequence selected fromthe group consisting of SEQ ID NO:1-5993.

In some embodiments, the purified population of bacteria areencapsulated in one or more of a polymer, carbohydrate, sugar, sugaralcohol, surfactant, plastic, glass, polysaccharide, lipid, wax, oil,fatty acid, amino acid, or glyceride. In some embodiment, theencapsulated bacteria are vitrified. In one embodiment, the encapsulatedbacteria are further encapsulated in a wax, fat, fatty acid, fattyalcohol, or glyceride.

In one embodiment, the purified population of bacteria are in the formof spores. In one embodiment, the spores are spray dried. In oneembodiment, the ruminant supplement is formulated as a tablet, capsule,pill, feed additive, food ingredient, food additive, food preparation,food supplement, consumable solution, consumable spray additive,consumable solid, consumable gel, injection, bolus, or combinationsthereof.

In some embodiments, the disclosure is drawn to a method of decreasingthe amount of carbon dioxide and/or carbonic acid in the rumen of aruminant, comprising: administering to a ruminant an effective amount ofa ruminant supplement comprising: a) a purified population of bacteriaselected from any one or more bacteria comprising a 16S nucleic acidsequence that is at least about 97% identical to any one of SEQ IDNO:1-5993; and b) a carrier suitable for ruminant administration;wherein the purified population of bacteria of a) is present in thesupplement in an amount effective to decrease the amount of carbondioxide and/or carbonic acid in the rumen of a ruminant administered thesupplement, as compared to a ruminant not administered the supplement.

In some embodiments, the disclosure is drawn to a method of decreasingthe amount of carbon dioxide and/or carbonic acid in the rumen of aruminant, comprising: administering to a ruminant an effective amount ofa ruminant supplement comprising: a) a purified population of bacteriaselected from: (i) Succinivibrio bacteria with a 16S nucleic acidsequence that is at least about 97% identical to SEQ ID NO:75, (ii)Prevotella bacteria with a 16S nucleic acid sequence that is at leastabout 97% identical to SEQ ID NO:86, and/or (iii) Bacteroides bacteriawith a 16S nucleic acid sequence that is at least about 97% identical toSEQ ID NO:13; and b) a carrier suitable for ruminant administration;wherein the purified population of bacteria of a) is present in thesupplement in an amount effective to decrease the amount of carbondioxide and/or carbonic acid in the rumen of a ruminant administered thesupplement, as compared to a ruminant not administered the supplement.

In one embodiment, at least one of the bacteria are selected from: (i)Succinivibrio bacteria with a 16S nucleic acid sequence that is at leastabout 97% identical to SEQ ID NO:75, (ii) Prevotella bacteria with a 16Snucleic acid sequence that is at least about 97% identical to SEQ IDNO:86, and/or (iii) Bacteroides bacteria with a 16S nucleic acidsequence that is at least about 97% identical to SEQ ID NO:13.

In one embodiment, the purified population of bacteria is selected from:(i) Succinivibrio bacteria with a 16S nucleic acid sequence that is atleast about 99% identical to SEQ ID NO:75, (ii)Prevotella bacteria witha 16S nucleic acid sequence that is at least about 99% identical to SEQID NO:86, and/or (iii) Bacteroides bacteria with a 16S nucleic acidsequence that is at least about 99% identical to SEQ ID NO:13.

In one embodiment, the purified population of bacteria is selected from:(i) Succinivibrio bacteria with a 16S nucleic acid sequence comprisingSEQ ID NO:75, (ii) Prevotella bacteria with a 16S nucleic acid sequencecomprising SEQ ID NO:86, and/or (iii) Bacteroides bacteria with a 16Snucleic acid sequence comprising SEQ ID NO:13.

In one embodiment, the purified population of bacteria is selected from:(i) Succinivibrio bacteria deposited as B-67550. (ii) Prevotellabacteria deposited as B-67552, and/or (iii) Bacteroides bacteriadeposited as B-67555.

In one embodiment, the ruminant supplement further comprises a purifiedpopulation of bacteria that comprises bacteria with a 16S nucleic acidsequence that is at least about 97% identical to a nucleic acid sequenceselected from the group consisting of SEQ ID NO:1-5993.

In one embodiment, the ruminant supplement further comprises a purifiedpopulation of bacteria that comprises bacteria with a 16S nucleic acidsequence that is at least about 99% identical to a nucleic acid sequenceselected from the group consisting of SEQ ID NO:1-5993.

In one embodiment, the ruminant supplement further comprises a purifiedpopulation of bacteria that comprises bacteria with a 16S nucleic acidsequence comprising a nucleic acid sequence selected from the groupconsisting of SEQ ID NO:1-5993.

In one embodiment, the purified population of bacteria are encapsulatedin one or more of a polymer, carbohydrate, sugar, sugar alcohol,surfactant, plastic, glass, polysaccharide, lipid, wax, oil, fatty acid,amino acid, or glyceride.

In one embodiment, the encapsulated bacteria are vitrified. In oneembodiment, the encapsulated bacteria are further encapsulated in a wax,fat, fatty acid, fatty alcohol, or glyceride. The method of claim 51,wherein the purified population of bacteria are in the form of spores.The method of claim 62, wherein the spores are spray dried.

In one embodiment, the ruminant supplement is formulated as a tablet,capsule, pill, feed additive, food ingredient, food additive, foodpreparation, food supplement, consumable solution, consumable sprayadditive, consumable solid, consumable gel, injection, bolus, orcombinations thereof.

In one embodiment, the ruminant is administered to a ruminant orally. Inone embodiment, the ruminant is a cow or a steer. In one embodiment, theruminant is fed a step-up diet. In one embodiment, the ruminant is fed afinishing diet.

In one embodiment, the disclosure is drawn to a ruminant supplementcapable of increasing the amount of meat marbling in a ruminant,comprising: a) a purified population of bacteria selected from any oneor more bacteria comprising a 16S nucleic acid sequence that is at leastabout 97% identical to any one of SEQ ID NO:1-5993: and b) a carriersuitable for ruminant administration; wherein the purified population ofbacteria of a) is present in the supplement in an amount effective toincrease the amount of meat marbling in the ruminant administered thesupplement, as compared to a ruminant not administered the supplement.

In one embodiment, the disclosure is drawn to a ruminant supplementcapable of increasing the amount of meat marbling in a ruminant,comprising: a) a purified population of bacteria selected from: (i)Succinivibrio bacteria with a 16S nucleic acid sequence that is at leastabout 97% identical to SEQ ID NO:75, (ii) Prevotella bacteria with a 16Snucleic acid sequence that is at least about 97% identical to SEQ IDNO:86, and/or (iii) Bacteroides bacteria with a 16S nucleic acidsequence that is at least about 97% identical to SEQ ID NO:13; and b) acarrier suitable for ruminant administration; wherein the purifiedpopulation of bacteria of a) is present in the supplement in an amounteffective to increase the amount of meat marbling in the ruminantadministered the supplement, as compared to a ruminant not administeredthe supplement.

In one embodiment, the least one of the bacteria are selected from: (i)Succinivibrio bacteria with a 16S nucleic acid sequence that is at leastabout 97% identical to SEQ ID NO:75, (ii) Prevotella bacteria with a 16Snucleic acid sequence that is at least about 97% identical to SEQ IDNO:86, and/or (iii) Bacteroides bacteria with a 16S nucleic acidsequence that is at least about 97% identical to SEQ ID NO:13.

In one embodiment, the purified population of bacteria is selected from:(i) Succinivibrio bacteria with a 16S nucleic acid sequence that is atleast about 99% identical to SEQ ID NO:75, (ii)Prevotella bacteria witha 16S nucleic acid sequence that is at least about 99% identical to SEQID NO:86, and/or (iii) Bacteroides bacteria with a 16S nucleic acidsequence that is at least about 99% identical to SEQ ID NO:13.

In one embodiment, the purified population of bacteria is selected from:(i) Succinivibrio bacteria with a 16S nucleic acid sequence comprisingSEQ ID NO:75, (ii) Prevotella bacteria with a 16S nucleic acid sequencecomprising SEQ ID NO:86, and/or (iii) Bacteroides bacteria with a 16Snucleic acid sequence comprising SEQ ID NO:13.

In one embodiment, the purified population of bacteria is selected from:(i) Succinivibrio bacteria deposited as B-67550, (ii) Prevotellabacteria deposited as B-67552, and/or (iii) Bacteroides bacteriadeposited as B-67555.

In one embodiment, the rumen supplement further comprises a purifiedpopulation of bacteria that comprises bacteria with a 16S nucleic acidsequence that is at least about 97% identical to a nucleic acid sequenceselected from the group consisting of SEQ ID NO:1-5993.

In one embodiment, the rumen supplement further comprises a purifiedpopulation of bacteria that comprises bacteria with a 16S nucleic acidsequence that is at least about 99% identical to a nucleic acid sequenceselected from the group consisting of SEQ ID NO:1-5993.

In one embodiment, the rumen supplement further comprising a purifiedpopulation of bacteria that comprises bacteria with a 16S nucleic acidsequence comprising a nucleic acid sequence selected from the groupconsisting of SEQ ID NO:1-5993.

In one embodiment, the purified population of bacteria are encapsulatedin one or more of a polymer, carbohydrate, sugar, sugar alcohol,surfactant, plastic, glass, polysaccharide, lipid, wax, oil, fatty acid,amino acid, or glyceride.

In one embodiment, the encapsulated bacteria are vitrified. In oneembodiment, the encapsulated bacteria are further encapsulated in a wax,fat, fatty acid, fatty alcohol, or glyceride. In one embodiment, thepurified population of bacteria are in the form of spores. In oneembodiment, the spores are spray dried.

In one embodiment, the rumen supplement is formulated as a tablet,capsule, pill, feed additive, food ingredient, food additive, foodpreparation, food supplement, consumable solution, consumable sprayadditive, consumable solid, consumable gel, injection, bolus, orcombinations thereof.

In some embodiments, the disclosure is drawn to a method increasing theamount of meat marbling in a ruminant, comprising: administering to aruminant an effective amount of a ruminant supplement comprising: a) apurified population of bacteria selected from any one or more bacteriacomprising a 16S nucleic acid sequence that is at least about 97%identical to any one of SEQ ID NO:1-5993; and b) a carrier suitable forruminant administration; wherein the purified population of bacteria ofa) is present in the supplement in an amount effective to increase theamount of meat marbling in the ruminant administered the supplement, ascompared to a ruminant not administered the supplement.

In some embodiments, the disclosure is drawn to a method increasing theamount of meat marbling in a ruminant, comprising: administering to aruminant an effective amount of a ruminant supplement comprising: a) apurified population of bacteria selected from: (i) Succinivibriobacteria with a 16S nucleic acid sequence that is at least about 97%identical to SEQ ID NO:75, (ii)Prevotella bacteria with a 16S nucleicacid sequence that is at least about 97% identical to SEQ ID NO:86,and/or (iii) Bacteroides bacteria with a 16S nucleic acid sequence thatis at least about 97% identical to SEQ ID NO:13; and b) a carriersuitable for ruminant administration; wherein the purified population ofbacteria of a) is present in the supplement in an amount effective toincrease the amount of meat marbling in the ruminant administered thesupplement, as compared to a ruminant not administered the supplement.

In one embodiment, at least one of the bacteria are selected from: (i)Succinivibrio bacteria with a 16S nucleic acid sequence that is at leastabout 97% identical to SEQ ID NO:75, (ii) Prevotella bacteria with a 16Snucleic acid sequence that is at least about 97% identical to SEQ IDNO:86, and/or (iii) Bacteroides bacteria with a 16S nucleic acidsequence that is at least about

97% identical to SEQ ID NO:13.

In one embodiment, the purified population of bacteria is selected from:(i) Succinivibrio bacteria with a 16S nucleic acid sequence that is atleast about 99% identical to SEQ ID NO:75, (ii) Prevotella bacteria witha 16S nucleic acid sequence that is at least about 99% identical to SEQID NO:86, and/or (iii) Bacteroides bacteria with a 16S nucleic acidsequence that is at least about

99% identical to SEQ ID NO:13.

In one embodiment, the purified population of bacteria is selected from:(i) Succinivibrio bacteria with a 16S nucleic acid sequence comprisingSEQ ID NO:75, (ii) Prevotella bacteria with a 16S nucleic acid sequencecomprising SEQ ID NO:86, and/or (iii) Bacteroides bacteria with a 16Snucleic acid sequence comprising SEQ ID NO:13.

In one embodiment, the purified population of bacteria is selected from:(i) Succinivibrio bacteria deposited as B-67550, (ii) Prevotellabacteria deposited as B-67552, and/or (iii) Bacteroides bacteriadeposited as B-67555.

In one embodiment, the ruminant supplement further comprises a purifiedpopulation of bacteria that comprises bacteria with a 16S nucleic acidsequence that is at least about 97% identical to a nucleic acid sequenceselected from the group consisting of SEQ ID NO:5993.

In one embodiment, the ruminant supplement further comprises a purifiedpopulation of bacteria that comprises bacteria with a 16S nucleic acidsequence that is at least about 99% identical to a nucleic acid sequenceselected from the group consisting of SEQ ID NO:5993.

In one embodiment, the ruminant supplement further comprises a purifiedpopulation of bacteria that comprises bacteria with a 16S nucleic acidsequence comprising a nucleic acid sequence selected from the groupconsisting of SEQ ID NO:1-5993.

In one embodiment, the purified population of bacteria are encapsulatedin one or more of a polymer, carbohydrate, sugar, sugar alcohol,surfactant, plastic, glass, polysaccharide, lipid, wax, oil, fatty acid,amino acid, or glyceride.

In one embodiment, the encapsulated bacteria are vitrified. In oneembodiment, the encapsulated bacteria are further encapsulated in a wax.On one embodiment, the purified population of bacteria are in the formof spores. In one embodiment, the spores are spray dried.

In one embodiment, the ruminant supplement is formulated as a tablet,capsule, pill, feed additive, food ingredient, food additive, foodpreparation, food supplement, consumable solution, consumable sprayadditive, consumable solid, consumable gel, injection, bolus, orcombinations thereof. In one embodiment, the ruminant supplement isadministered to a ruminant orally. In one embodiment, the ruminant is acow or a steer. In one embodiment, the ruminant is fed a step-up diet.In one embodiment the ruminant is fed a finishing diet. In oneembodiment, the increase in meat marbling is an increase of at least10%.

In some embodiments, the disclosure is drawn to a ruminant supplementcapable of increasing feed efficiency in a ruminant, comprising: a) apurified population of bacteria selected from any one or more bacteriacomprising a 16S nucleic acid sequence that is at least about 97%identical to any one of SEQ ID NO:1-5993: and b) a carrier suitable forruminant administration; wherein the purified population of bacteria ofa) is present in the supplement in an amount effective to increase feedefficiency in a ruminant administered the supplement, as compared to aruminant not administered the supplement.

In some embodiments, the disclosure is drawn to a ruminant supplementcapable of increasing feed efficiency in a ruminant, comprising: a) apurified population of bacteria selected from: (i) Succinivibriobacteria with a 16S nucleic acid sequence that is at least about 97%identical to SEQ ID NO:75, (ii) Prevotella bacteria with a 16S nucleicacid sequence that is at least about 97% identical to SEQ ID NO:86,and/or (iii) Bacteroides bacteria with a 16S nucleic acid sequence thatis at least about 97% identical to SEQ ID NO:13; and b) a carriersuitable for ruminant administration; wherein the purified population ofbacteria of a) is present in the supplement in an amount effective toincrease feed efficiency in a ruminant administered the supplement, ascompared to a ruminant not administered the supplement.

In some embodiments, the at least one of the bacteria are selected from:(i) Succinivibrio bacteria with a 16S nucleic acid sequence that is atleast about 97% identical to SEQ ID NO:75, (ii)Prevotella bacteria witha 16S nucleic acid sequence that is at least about 97% identical to SEQID NO:86, and/or (iii) Bacteroides bacteria with a 16S nucleic acidsequence that is at least about 97% identical to SEQ ID NO:13.

In some embodiments, the disclosure is drawn to a method for increasingfeed efficiency in a ruminant, comprising: administering to a ruminantan effective amount of a ruminant supplement comprising: a) a purifiedpopulation of bacteria selected from any one or more bacteria comprisinga 16S nucleic acid sequence that is at least about 97% identical to anyone of SEQ ID NO:1-5993; and b) a carrier suitable for ruminantadministration; wherein the purified population of bacteria of a) ispresent in the supplement in an amount effective to increase feedefficiency in a ruminant administered the supplement, as compared to aruminant not administered the supplement.

In some embodiments, the method increasing feed efficiency in a ruminantcomprises: administering to a ruminant an effective amount of a ruminantsupplement comprising: a) a purified population of bacteria selectedfrom: (i) Succinivibrio bacteria with a 16S nucleic acid sequence thatis at least about 97% identical to SEQ ID NO:75, (ii) Prevotellabacteria with a 16S nucleic acid sequence that is at least about 97%identical to SEQ ID NO:86, and/or (iii) Bacteroides bacteria with a 16Snucleic acid sequence that is at least about 97% identical to SEQ IDNO:13; and b) a carrier suitable for ruminant administration; whereinthe purified population of bacteria of a) is present in the supplementin an amount effective to increase feed efficiency in a ruminantadministered the supplement, as compared to a ruminant not administeredthe supplement.

In some embodiments, the at least one of the bacteria are selected from:(i) Succinivibrio bacteria with a 16S nucleic acid sequence that is atleast about 97% identical to SEQ ID NO:75, (ii) Prevotella bacteria witha 16S nucleic acid sequence that is at least about 97% identical to SEQID NO:86, and/or (iii) Bacteroides bacteria with a 16S nucleic acidsequence that is at least about 97% identical to SEQ ID NO:13.

In some embodiments, the ruminant supplement capable of increasingperformance in a ruminant, comprising: a) a purified population ofbacteria selected from any one or more bacteria comprising a 16S nucleicacid sequence that is at least about 97% identical to any one of SEQ IDNO:1-5993: and b) a carrier suitable for ruminant administration;wherein the purified population of bacteria of a) is present in thesupplement in an amount effective to increase performance in a ruminantadministered the supplement, as compared to a ruminant not administeredthe supplement.

In some embodiments, the ruminant supplement capable of increasingperformance in a ruminant, comprising: a) a purified population ofbacteria selected from: (i) Succinivibrio bacteria with a 16S nucleicacid sequence that is at least about 97% identical to SEQ ID NO:75, (ii)Prevotella bacteria with a 16S nucleic acid sequence that is at leastabout 97% identical to SEQ ID NO:86, and/or (iii) Bacteroides bacteriawith a 16S nucleic acid sequence that is at least about 97% identical toSEQ ID NO:13; and b) a carrier suitable for ruminant administration;wherein the purified population of bacteria of a) is present in thesupplement in an amount effective to increase performance in a ruminantadministered the supplement, as compared to a ruminant not administeredthe supplement.

In some embodiments, at least one of the bacteria are selected from: (i)Succinivibrio bacteria with a 16S nucleic acid sequence that is at leastabout 97% identical to SEQ ID NO:75, (ii)Prevotella bacteria with a 16Snucleic acid sequence that is at least about 97% identical to SEQ IDNO:86, and/or (iii) Bacteroides bacteria with a 16S nucleic acidsequence that is at least about 97% identical to SEQ ID NO:13.

In some embodiments, the disclosure is drawn to a ruminant supplementcapable of reducing methane production and emission in a ruminant,comprising: a) a purified population of bacteria selected from any oneor more bacteria comprising a 16S nucleic acid sequence that is at leastabout 97% identical to any one of SEQ ID NO:1-5993: and b) a carriersuitable for ruminant administration; wherein the purified population ofbacteria of a) is present in the supplement in an amount effective toreduce methane production and emission in a ruminant administered thesupplement, as compared to a ruminant not administered the supplement.

In some embodiments, the disclosure is drawn to a ruminant supplementcapable of reducing methane production and emission in a ruminant,comprising: a) a purified population of bacteria selected from: (i)Succinivibrio bacteria with a 16S nucleic acid sequence that is at leastabout 97% identical to SEQ ID NO:75, (ii) Prevotella bacteria with a 16Snucleic acid sequence that is at least about 97% identical to SEQ IDNO:86, and/or (iii) Bacteroides bacteria with a 16S nucleic acidsequence that is at least about 97% identical to SEQ ID NO:13; and b) acarrier suitable for ruminant administration; wherein the purifiedpopulation of bacteria of a) is present in the supplement in anamounteffective to reduce methane production and emission in a ruminantadministered the supplement, as compared to a ruminant not administeredthe supplement.

In some embodiments, at least one of the bacteria are selected from: (i)Succinivibrio bacteria with a 16S nucleic acid sequence that is at leastabout 97% identical to SEQ ID NO:75, (ii)Prevotella bacteria with a 16Snucleic acid sequence that is at least about 97% identical to SEQ IDNO:86, and/or (iii) Bacteroides bacteria with a 16S nucleic acidsequence that is at least about 97% identical to SEQ ID NO:13.

In some embodiments, the disclosure is drawn to a ruminant supplementcapable of reducing methane production and emission in a ruminant,comprising: a) a purified population of bacteria selected from any oneor more bacteria comprising a 16S nucleic acid sequence that is at leastabout 97% identical to any one of SEQ ID NO:1-5993: and b) a carriersuitable for ruminant administration; wherein the purified population ofbacteria of a) is present in the supplement in an amount effective toreduce methane production and emission in a ruminant administered thesupplement, as compared to a ruminant not administered the supplement.

In some embodiments, the disclosure is drawn to a ruminant supplementcapable of reducing methane production and emission in a ruminant,comprising: a) a purified population of bacteria selected from: (i)Succinivibrio bacteria with a 16S nucleic acid sequence that is at leastabout 97% identical to SEQ ID NO:75, (ii) Prevotella bacteria with a 16Snucleic acid sequence that is at least about 97% identical to SEQ IDNO:86, and/or (iii) Bacteroides bacteria with a 16S nucleic acidsequence that is at least about 97% identical to SEQ ID NO:13; and b) acarrier suitable for ruminant administration; wherein the purifiedpopulation of bacteria of a) is present in the supplement in an amounteffective to reduce methane production and emission in a ruminantadministered the supplement, as compared to a ruminant not administeredthe supplement.

In some embodiments, the at least one of the bacteria are selected from:(i) Succinivibrio bacteria with a 16S nucleic acid sequence that is atleast about 97% identical to SEQ ID NO:75, (ii)Prevotella bacteria witha 16S nucleic acid sequence that is at least about 97% identical to SEQID NO:86, and/or (iii) Bacteroides bacteria with a 16S nucleic acidsequence that is at least about 97% identical to SEQ ID NO:13.

In some embodiments, the microbes are administered with a prebiotic, avitamin, or a mineral. In some embodiments, the microbes areadministered with vitamin B or a precursor thereof.

Budapest Treaty on the International Recognition of the Deposit ofMicroorganisms for the Purpose of Patent Procedures

The microorganisms described in this application were deposited with (1)the American Type Culture Collection (ATCC®), located at 10801University Blvd., Manassas, Va. 20110, USA; and the United StatesDepartment of Agriculture (USDA) Agricultural Research Service (ARS)Culture Collection (NRRL®), located at 1815 N. University St., Peoria,Ill. 61604, USA

The deposits were made under the terms of the Budapest Treaty on theInternational Recognition of the Deposit of Microorganisms for thePurposes of Patent Procedure. The ATCC and NRRL accession numbers forthe aforementioned Budapest Treaty deposits are provided in Table 1. TheAccession numbers and corresponding dates of deposit for themicroorganisms described in this application are separately provided inTable 2.

The strains designated in the below table have been deposited in thelabs of Ascus Biosciences, Inc. since at least Apr. 22, 2017, and August2017.

In Table 1, the closest predicted hits for taxonomy of the microbes arelisted in columns 2, and 5. Column 2 is the top taxonomic hit predictedby BLAST, and column 5 is the top taxonomic hit for genus+speciespredicted by BLAST. The strains designated in the below table have beendeposited in the labs of Ascus Biosciences, Inc. since at least Apr. 22,2017, and August, 2017.

TABLE 1 Microbes of the present disclosure, including bacteria (1-190).BLAST Taxonomic BLAST Sequence Identifier Predicted Closest Taxa of TopHit w/Genus + % Query Strain for Associated MIC Isolated MicrobesSpecies Ident. Cover Designation Marker Score 1. Prevotella (genus)Prevotella ruminicola 93%  98% Ascusbbf_6176 SEQ ID NO: 1 1 2.Prevotella (genus) Prevotella loescheii 88%  99% Ascusbbf_22143 SEQ IDNO: 2 1 3. Prevotella (genus) Prevotella ruminicola 91% 100%Ascusbbf_4883 SEQ ID NO: 3 0.97095 4. Selenomonas (genus) Selenomonas93%  95% Ascusbbf_13543 SEQ ID NO: 4 0.97095 ruminantium 5. ClostridiumXIVa Oscillibacter 92% 100% Ascusbbf_152 SEQ ID NO: 5 0.88129 (Cluster)valericigenes 6. Clostridium XIVa Oscillibacter Ascusbbf_152A SEQ ID NO:5398 0.88129 (Cluster) valericigenes 7. Prevotella (genus) Prevotellaruminicola 94% 100% Ascusbbf_707 SEQ ID NO: 6 0.88129 8. Fibrobacter(genus) Fibrobacter 99% 100% Ascusbbf_1238 SEQ ID NO: 7 0.88129intestinalis 9. Prevotella (genus) Prevotella ruminicola 89% 100%Ascusbbf_5588 SEQ ID NO: 8 0.88129 10. SaccharofermentansSaccharofermentans 86% 100% Ascusbbf_4691 SEQ ID NO: 9 0.88129 (genus)acetigenes 11. Saccharofermentans Saccharofermentans Ascusbbf_4691C SEQID NO: 5425 0.88129 (genus) acetigenes 12. SaccharofermentansIntestinimonas 86% 100% Ascusbbf_59499 SEQ ID NO: 10 0.88129 (genus)butyriciproducens 13. Bacillus (genus) Brevibacillus brevis 86%  89%Ascusbbf_9770 SEQ ID NO: 11 0.88129 14. Spirochaeta (genus) Treponemaparvum 88%  92% Ascusbbf_123632 SEQ ID NO: 12 0.88129 15. Bacteroides(genus) Bacteroides 99%  97% Ascusbbf_14146 SEQ ID NO: 13 0.78606xylanisolvens 16. Lachnospiracea incertae Desulfotomaculum sp. 94% 100%Ascusbbf_1103 SEQ ID NO: 14 0.67032 sedis (genus) 17. Clostridium XIVaLachnoclostridium 89% 100% Ascusbbf_498 SEQ ID NO: 15 0.66823 (Cluster)pacaense 18. Prevotella (genus) Prevotella oralis 89% 100%Ascusbbf_13717 SEQ ID NO: 16 0.65415 19. Prevotella (genus) Prevotellaoralis Ascusbbf_13717A SEQ ID NO: 5450 0.65415 20. Clostridium XIVaCoprococcus catus 90%  97% Ascusbbf_876 SEQ ID NO: 17 0.65106 (Cluster)21. Bacteroides (genus) Bacteroides uniformis 89% 100% Ascusbbf_612 SEQID NO: 18 0.65002 22. Selenomonas (genus) Selenomonas 97% 100%Ascusbbf_4936 SEQ ID NO: 19 0.63816 ruminantium 23. Selenomonas (genus)Selenomonas Ascusbbf_4936A SEQ ID NO: 5460 0.63816 ruminantium 24.Prevotella (genus) Prevotella oulorum 92% 100% Ascusbbf_6809 SEQ ID NO:20 0.6337 25. Clostridium XIVa Clostridium 91% 100% Ascusbbf_113152 SEQID NO: 21 0.63008 (Cluster) aminophilum 26. Clostridium XIVa ClostridiumAscusbbf_113152A SEQ ID NO: 5462 0.63008 (Cluster) aminophilum 27.Ruminococcus (genus) Ruminococcus bromii 99%  96% Ascusbbf_18 SEQ ID NO:22 0.62713 28. Prevotella (genus) Prevotella ruminicola 94% 100%Ascusbbf_9031 SEQ ID NO: 23 0.62075 29. Spirochaeta (genus) Treponema86%  98% Ascusbbf_11823 SEQ ID NO: 24 0.61287 brennaborense 30.Butyricimonas (genus) Porphyromonadaceae 87%  98% Ascusbbf_1007 SEQ IDNO: 25 0.60495 31. Butyricimonas (genus) PorphyromonadaceaeAscusbbf_1007A SEQ ID NO: 5473 0.60495 32. Prevotella (genus) Prevotellabaroniae 87%  99% Ascusbbf_24422 SEQ ID NO: 26 0.59156 33. Prevotella(genus) Prevotella baroniae Ascusbbf_24422A SEQ ID NO: 5474 0.59156 34.Olsenella (genus) Olsenella umbonata 99% 100% Ascusbbf_951 SEQ ID NO: 270.59007 35. Clostridium XIVa [Clostridium] 96% 100% Ascusbbf_80169 SEQID NO: 28 0.58852 (Cluster) symbiosum 36. Spirochaeta (genus) Treponemabryantii 90%  97% Ascusbbf_5699 SEQ ID NO: 29 0.58423 37. Spirochaeta(genus) Treponema bryantii Ascusbbf_5699B SEQ ID NO: 5483 0.58423 38.Prevotella (genus) Prevotella ruminicola 91% 100% Ascusbbf_130 SEQ IDNO: 30 0.58333 39. Acidaminococcus (genus) Acidaminococcus 95% 100%Ascusbbf_10109 SEQ ID NO: 31 0.58267 fermentans 40. Parabacteroides(genus) Culturomica 86%  89% Ascusbbf_29797 SEQ ID NO: 32 0.58241massiliensis 41. Parabacteroides (genus) Culturomica Ascusbbf_29797A SEQID NO: 5502 0.58241 massiliensis 42. Clostridium sensu strictoChristensenella 86%  99% Ascusbbf_24410 SEQ ID NO: 33 0.58142 (genus)timonensis 43. Oribacterium (genus) Oribacterium sinus 91%  99%Ascusbbf_54068 SEQ ID NO: 34 0.58113 44. Clostridium XIVa [Clostridium]bolteae 93% 100% Ascusbbf_7003 SEQ ID NO: 35 0.58059 (Cluster) 45.Pseudoflavonifractor Intestinimonas 89% 100% Ascusbbf_23 SEQ ID NO: 360.57785 (genus) butyriciproducens 46. Prevotella (genus) Prevotellaruminicola 90% 100% Ascusbbf_1697 SEQ ID NO: 37 0.57337 47. Prevotella(genus) Prevotella ruminicola Ascusbbf_1697B SEQ ID NO: 5511 0.57337 48.Treponema (genus) Treponema zioleckii 99%  99% Ascusbbf_24513 SEQ ID NO:38 0.5696 49. Prevotella (genus) Prevotella oralis 89% 100%Ascusbbf_7586 SEQ ID NO: 39 0.56896 50. Butyricimonas (genus)Barnesiella viscericola 85%  91% Ascusbbf_27854 SEQ ID NO: 40 0.5665751. Saccharofermentans Oscillibacter 87% 100% Ascusbbf_1034 SEQ ID NO:41 0.56476 (genus) valericigenes 52. Saccharofermentans OscillibacterAscusbbf_1034A SEQ ID NO: 5517 0.56476 (genus) valericigenes 53.Butyricimonas (genus) Butyricimonas virosa 82% 100% Ascusbbf_23134 SEQID NO: 42 0.56219 54. Butyricimonas (genus) Butyricimonas virosaAscusbbf_23134A SEQ ID NO: 5519 0.56219 55. Rhodobacter (genus)Gemmobacter 99%  87% Ascusbbf_7027 SEQ ID NO: 43 0.56127 intermedius 56.Prevotella (genus) Butyricimonas virosa 84%  92% Ascusbbf_43679 SEQ IDNO: 44 0.56056 57. Fluviicola (genus) Anaerocella delicata 85%  87%Ascusbbf_63954 SEQ ID NO: 45 0.55952 58. Fluviicola (genus) Anaerocelladelicata Ascusbbf_63954A SEQ ID NO: 5526 0.55952 59. Succiniclasticum(genus) Succiniclasticum 95%  95% Ascusbbf_1517 SEQ ID NO: 46 0.55908ruminis 60. Solobacterium (genus) Solobacterium moorei 91%  99%Ascusbbf_104 SEQ ID NO: 47 0.55759 61. Clostridium XIVa [Clostridium]90% 100% Ascusbbf_148 SEQ ID NO: 48 0.55551 (Cluster) lavalense 62.Prevotella (genus) Prevotella bryantii 99% 100% Ascusbbf_944 SEQ ID NO:49 0.55265 63. Lachnospiracea incertae Eubacterium 90% 100%Ascusbbf_76009 SEQ ID NO: 50 0.55253 sedis (genus) oxidoreducens 64.Veillonella (genus) Holdemania filiformis 84%  96% Ascusbbf_23033 SEQ IDNO: 51 0.55253 65. Cellulosimicrobium Cellulosimicrobium 95% 100%Ascusbbf_20389 SEQ ID NO: 52 0.55131 (genus) cellulans 66. Cupriavidus(genus) Sutterella 92% 100% Ascusbbf_2600 SEQ ID NO: 53 0.54892wadsworthensis 67. Bacteroides (genus) Paraprevotella 86%  92%Ascusbbf_8118 SEQ ID NO: 54 0.54888 xylaniphila 68. Prevotella (genus)Prevotella ruminicola 92% 100% Ascusbbf_201 SEQ ID NO: 55 0.54656 69.Prevotella (genus) Prevotella ruminicola Ascusbbf_20IK SEQ ID NO: 55760.54656 70. Spirochaeta (genus) Treponema 88% 100% Ascusbbf_6315 SEQ IDNO: 56 0.54535 saccharophilum 71. Megasphaera (genus) Megasphaeraelsdenii 99% 100% Ascusbbf_10712 SEQ ID NO: 57 0.54494 72. Megasphaera(genus) Megasphaera elsdenii Ascusbbf_10712E SEQ ID NO: 5582 0.54494 73.Succinivibrio (genus) Succinivibrio 90%  99% Ascusbbf_6012 SEQ ID NO: 580.54428 dextrinosolvens 74. Succinivibrio (genus) SuccinivibrioAscusbbf_6012C SEQ ID NO: 5589 0.54428 dextrinosolvens 75. Spirochaeta(genus) Treponema bryantii 98%  99% Ascusbbf_2297 SEQ ID NO: 59 0.5441376. Spirochaeta (genus) Treponema bryantii Ascusbbf_2297G SEQ ID NO:5598 0.54413 77. Bacteroides (genus) Bacteroides uniformis 89% 100%Ascusbbf_9540 SEQ ID NO: 60 0.54383 78. Oscillibacter (genus)Oscillibacter 94% 100% Ascusbbf_873 SEQ ID NO: 61 0.54374 valericigenes79. Prevotella (genus) Prevotella dentalis 83%  95% Ascusbbf_87102 SEQID NO: 62 0.54356 80. Pseudomonas (genus) Pseudomonas 98%  99%Ascusbbf_77105 SEQ ID NO: 63 0.54356 pertucinogena 81. Corynebacterium(genus) Corynebacterium 99% 100% Ascusbbf_269 SEQ ID NO: 64 0.54206marinum 82. Adlercreutzia (genus) Raoultibacter 92% 100% Ascusbbf_41015SEQ ID NO: 65 0.54192 massiliensis 83. Adlercreutzia (genus)Raoultibacter Ascusbbf_41015A SEQ ID NO: 5614 0.54192 massiliensis 84.Acidaminococcus (genus) Acidaminococcus 98%  97% Ascusbbf_32877 SEQ IDNO: 66 0.54166 fermentans 85. Acidaminococcus (genus) AcidaminococcusAscusbbf_32877A SEQ ID NO: 5619 0.54166 fermentans 86. Dorea (genus)Dorea longicatena 99% 100% Ascusbbf_57294 SEQ ID NO: 67 0.53443 87.Dorea (genus) Dorea longicatena Ascusbbf_57294B SEQ ID NO: 5621 0.5344388. Roseburia (genus) Howardella ureilytica 88%  98% Ascusbbf_27932 SEQID NO: 68 0.53375 89. Anaerovibrio (genus) Anaerovibrio 95%  97%Ascusbbf_22558 SEQ ID NO: 69 0.53353 lipolyticus 90. Anaerovibrio(genus) Anaerovibrio Ascusbbf_22558B SEQ ID NO: 5627 0.53353 lipolyticus91. Bacteroides (genus) Bacteroides 88% 100% Ascusbbf_983757 SEQ ID NO:70 0.5317 helcogenes 92. Bacteroides (genus) BacteroidesAscusbbf_983757B SEQ ID NO: 5629 0.5317 helcogenes 93. Clostridium XIVaClostridium 98% 100% Ascusbbf_52330 SEQ ID NO: 71 0.53133 (Cluster)aminophilum 94. Clostridium XIVa Clostridium Ascusbbf_52330A SEQ ID NO:5631 0.53133 (Cluster) aminophilum 95. Sporosarcina (genus)Lactobacillus floricola 79%  97% Ascusbbf_88445 SEQ ID NO: 72 0.5306996. Streptomyces (genus) Streptomyces albus 99% 100% Ascusbbf_4111 SEQID NO: 73 0.53006 97. Syntrophococcus (genus) Syntrophococcus 93% 100%Ascusbbf_1085 SEQ ID NO: 74 0.5294 sucromutans 98. Succinivibrio (genus)Succinivibrio 99%  99% Ascusbbf_154 SEQ ID NO: 75 0.52737dextrinosolvens 99. Selenomonas (genus) Selenomonas bovis 99% 100%Ascusbbf_1010 SEQ ID NO: 76 0.527 100. Parabacteroides Megasphaeraindica 99%  99% Ascusbbf_5575 SEQ ID NO: 77 0.52675 (genus) 101.Parabacteroides Megasphaera indica Ascusbbf_5575B SEQ ID NO: 56630.52675 (genus) 102. Prevotella (genus) Prevotella oris 82% 100%Ascusbbf_775 SEQ ID NO: 78 0.52672 103. Prevotella (genus) Prevotellaoris Ascusbbf_775A SEQ ID NO: 5670 0.52672 104. Butyrivibrio (genus)Butyrivibrio 96% 100% Ascusbbf_19348 SEQ ID NO: 79 0.52608 fibrisolvens105. Clostridium sensu Clostridium 99% 100% Ascusbbf_24302 SEQ ID NO: 800.52361 stricto (genus) beijerinckii 106. Succinivibrio (genus)Succinivibrio 99%  97% Ascusbbf_1 SEQ ID NO: 81 0.51924 dextrinosolvens107. Lachnobacterium Lachnobacterium 99%  99% Ascusbbf_52548 SEQ ID NO:82 0.51683 (genus) bovis 108. Clostridium IV Clostridiales bacterium 93%100% Ascusbbf_50658 SEQ ID NO: 83 0.51263 (Cluster) 109. LachnospiraceaLachnospira 89% 100% Ascusbbf_850 SEQ ID NO: 84 0.5088 incertae sedis(genus) pectinoschiza 110. Parabacteroides Parabacteroides 84% 100%Ascusbbf_25259 SEQ ID NO: 85 0.50691 (genus) distasonis 111. Prevotella(genus) Prevotella albensis 98% 100% Ascusbbf_4 SEQ ID NO: 86 0.50464112. Bacteroides (genus) Bacteroides uniformis 89% 100% Ascusbbf_5131SEQ ID NO: 87 0.49238 113. Clostridium IV Caproiciproducens 89%  95%Ascusbbf_8600 SEQ ID NO: 88 0.47814 (Cluster) galactitolivorans 114.Clostridium IV Caproiciproducens Ascusbbf_8600B SEQ ID NO: 5726 0.47814(Cluster) galactitolivorans 115. Pyramidobacter Rarimicrobium 92%  94%Ascusbbf_1273 SEQ ID NO: 89 0.46972 (genus) hominis 116. Ruminococcus(genus) Ruminococcus 98%  99% Ascusbbf_39159 SEQ ID NO: 90 0.46727flavefaciens 117. Coprococcus (genus) Eubacterium 89% 100% Ascusbbf_9751SEQ ID NO: 91 0.4618 oxidoreducens 118. Ruminobacter (genus)Ruminobacter 99% 100% Ascusbbf_318 SEQ ID NO: 92 0.45953 amylophilus119. Thermobifida (genus) Thermobifida fusca 99% 100% Ascusbbf_7046 SEQID NO: 93 0.45752 120. Papillibacter (genus) Oscillibacter 86% 100%Ascusbbf_25993 SEQ ID NO: 94 0.45023 valericigenes 121. Rhodobacter(genus) Rhodobacter 95%  99% Ascusbbf_7027 SEQ ID NO: 95 0.56127gluconicum 122. Prevotella (genus) Gabonibacter 87%  76%Ascusbbf_1372985 SEQ ID NO: 96 0.55953 massiliensis 123. Prevotella(genus) Gabonibacter Ascusbbf_1372985F SEQ ID NO: 5746 0.55953massiliensis 124. Aquamarina atlantica Gabonibacter 89%  76%Ascusbbf_23253 SEQ ID NO: 97 0.50683 (genus + species) massiliensis 125.Aquamarina pacifica Gabonibacter 86%  87% Ascusbbf_121971 SEQ ID NO: 980.42 (genus + species) massiliensis 126. Aquamarina pacificaGabonibacter Ascusbbf_121971A SEQ ID NO: 5757 0.42 (genus + species)massiliensis 127. Treponema bryantii Treponema bryantii 98%  94%Ascusbbf_5251 SEQ ID NO: 99 0.56738 (genus + species) 128. Treponemabryantii Treponema bryantii Ascusbbf_5251G SEQ ID NO: 5764 0.56738(genus + species) 129. Actinomyces turicensis Actinomyces turicensis 85%100% Ascusbbf_6716 SEQ ID NO: 100 0.5201 (genus + species) 130.Prevotella (genus) Prevotella oulorum 92%  99% Ascusbbf_100 SEQ ID NO:101 0.53729 131. Staphylococcus Paenibacillus 84%  84% Ascusbbf_20584SEQ ID NO: 102 0.52104 (genus) hemerocallicola 132. Prevotella (genus)Prevotella ruminicola 88% 100% Ascusbbf_4317 SEQ ID NO: 103 0.55564 133.Prevotella (genus) Prevotella ruminicola Ascusbbf_4317D SEQ ID NO: 57770.55564 134. Prevotella (genus) Prevotella ruminicola 90%  93%Ascusbbf_6 SEQ ID NO: 104 0.46763 135. Mogibacterium Mogibacterium 91%100% Ascusbbf_19022 SEQ ID NO: 105 0.47803 (genus) pumilum 136.Pseudobutyribibrio Pseudobutyrivibrio 99% 100% Ascusbbf_2624 SEQ ID NO:106 0.52337 (genus) ruminis 137. Pseudobutyribibrio PseudobutyrivibrioAscusbbf_2624D SEQ ID NO: 5797 0.52337 (genus) ruminis 138. Fluviicola(genus) Fluviicola taffensis 84%  90% Ascusbbf_3427 SEQ ID NO: 1070.50515 139. Fluviicola (genus) Fluviicola taffensis Ascusbbf_3427B SEQID NO: 5802 0.50515 140. Prevotella (genus) Prevotella ruminicola 92%100% Ascusbbf_5005 SEQ ID NO: 108 0.57034 141. Prevotella (genus)Prevotella ruminicola 100%  100% Ascusbbf_69 SEQ ID NO: 109 0.50536 142.Succiniclasticum Succiniclasticum 90%  90% Ascusbbf_8082 SEQ ID NO: 1100.50084 (genus) ruminis 143. Prevotella (genus) Prevotella ruminicola94% 100% Ascusbbf_95 SEQ ID NO: 111 0.53509 144. Clostridium XIVaButyrivibrio 89% 100% Ascusbbf_1136 SEQ ID NO: 112 0.50966 (cluster)fibrisolvens 145. Asteroleplasma Asteroleplasma 98% 100% Ascusbbf_2770SEQ ID NO: 113 0.51006 (genus) anaerobium 146. Turicibacter (genus)Turicibacter sanguinis 98% 100% Ascusbbf_1629 SEQ ID NO: 114 0.51632147. Prevotella (genus) Bacteroides caecicola 85%  99% Ascusbbf_1821 SEQID NO: 115 0.53784 148. Prevotella (genus) Prevotella ruminicola 95%100% Ascusbbf_56782 SEQ ID NO: 116 0.5317 149. Olsenella (genus)Olsenella scatoligenes 99% 100% Ascusbbf_92 SEQ ID NO: 117 0.46089 150.Prevotella (genus) Prevotella ruminicola 94%  99% Ascusbbf_118 SEQ IDNO: 118 0.6108 151. Prevotella (genus) Prevotella ruminicolaAscusbbf_118B SEQ ID NO: 5868 0.6108 152. Aggregatibacter Prevotellaruminicola 87%  44% Ascusbbf_5429 SEQ ID NO: 119 0.57983 (genus) 153.Aggregatibacter Prevotella ruminicola Ascusbbf_5429C SEQ ID NO: 58720.57983 (genus) 154. Ruminobacter (genus) Ruminobacter 86%  88%Ascusbbf_3 SEQ ID NO: 120 0.56323 amylophilus 155. Prevotella (genus)Prevotella ruminicola 91%  99% Ascusbbf_10576 SEQ ID NO: 121 0.56208156. Prevotella (genus) Prevotella ruminicola 93%  98% Ascusbbf_729 SEQID NO: 122 0.54949 157. Prevotella (genus) Prevotella ruminicola 92%100% Ascusbbf_201 SEQ ID NO: 123 0.54656 158. Prevotella (genus)Prevotella ruminicola 91%  99% Ascusbbf_416 SEQ ID NO: 124 0.53816 159.Prevotella (genus) Prevotella ruminicola 94%  99% Ascusbbf_15806 SEQ IDNO: 125 0.52527 160. Clostridium XIVa Clostridium 94% 100% Ascusbbf_6115SEQ ID NO: 126 0.52278 (cluster) aminophilum 161. Anaerovibrio (genus)Anaerovibrio 95%  97% Ascusbbf_1325058 SEQ ID NO: 127 0.52183lipolyticus 162. Prevotella (genus) Prevotella buccalis 91% 100%Ascusbbf_28350 SEQ ID NO: 128 0.51744 163. Parabacteroides Muribaculum93% 100% Ascusbbf_372 SEQ ID NO: 129 0.51572 (genus) intestinale 164.Phascolarctobacterium Phascolarctobacterium 96%  93% Ascusbbf_667 SEQ IDNO: 130 0.51381 (genus) succinatutens 165. PhascolarctobacteriumPhascolarctobacterium Ascusbbf_667A SEQ ID NO: 5930 0.51381 (genus)succinatutens 166. Bacteroides (genus) Bacteroides 83% 100%Ascusbbf_1207 SEQ ID NO: 131 0.51075 coprophilus 167. LachnospiraceaCoprococcus catus 92%  97% Ascusbbf_3875 SEQ ID NO: 132 0.47237 incertaesedis (genus) 168. Clostridium XIVa Clostridium 90% 100% Ascusbbf_72889SEQ ID NO: 133 0.46531 (cluster) aminophilum 169. Clostridium XIVaClostridium Ascusbbf_72889B SEQ ID NO: 5947 0.46531 (cluster)aminophilum 170. Parabacteroides Barnesiella viscericola 85%  94%Ascusbbf_106863 SEQ ID NO: 134 0.45152 (genus) 171. ParabacteroidesBarnesiella viscericola Ascusbbf_106863B SEQ ID NO: 5949 0.45152 (genus)172. Prevotella (genus) Prevotella ruminicola 94%  97% Ascusbbf_120 SEQID NO: 135 0.53376 173. Prevotella (genus) Prevotella ruminicola 93% 99% Ascusbbf_930 SEQ ID NO: 136 0.51321 174. Bacteroides (genus)Bacteroides uniformis 89% 100% Ascusbbf_915 SEQ ID NO: 5369 0.54396 175.Bacteroides (genus) Bacteroides uniformis Ascusbbf_915A SEQ ID NO: 59550.54396 176. Prevotella (genus) Prevotella ruminicola 88%  98%Ascusbbf_8941 SEQ ID NO: 5370 0.5328 177. Prevotella (genus) Prevotellaruminicola Ascusbbf_8941A SEQ ID NO: 5956 0.5328 178. AnaerovobrioAnaerovibrio 96%  97% Ascusbbf_8480 SEQ ID NO: 5371 0.48193 lipolyticus179. Anaerovobrio Anaerovibrio Ascusbbf_8480A SEQ ID NO: 5989 0.48193lipolyticus 180. Prevotella (genus) Prevotella ruminicola 92%  98%Ascusbbf_374 SEQ ID NO: 5372 0.52368 181. Prevotella (genus) Prevotellaruminicola Ascusbbf_374C SEQ ID NO: 5991 0.52368 182. Prevotella (genus)Prevotella brevis 92% 100% Ascusbbf_6906 SEQ ID NO: 5373 0.4889 183.Prevotella (genus) Prevotella ruminicola 94%  99% Ascusbbf_721 SEQ IDNO: 5374 0.88129 184. Syntrophococcus Syntrophococcus 93% 100%Ascusbbf_3819 SEQ ID NO: 5375 0.45798 (genus) sucromutans 185.Syntrophococcus Syntrophococcus Ascusbbf_3819A SEQ ID NO: 5971 0.45798(genus) sucromutans 186. Bacteroides (genus) Bacteroides coprocola 87%100% Ascusbbf_4323 SEQ ID NO: 5376 0.50634 187. Bacteroides (genus)Bacteroides coprocola Ascusbbf_4323B SEQ ID NO: 5973 0.50634 188.Prevotella (genus) Prevotella ruminicola 91% 100% Ascusbbf_6087 SEQ IDNO: 5377 0.52954 189. Prevotella (genus) Prevotella ruminicolaAscusbbf_6087B SEQ ID NO: 5977 0.52954 190. SaccharofermentansChristensenella 86%  99% Ascusbbf_8414 SEQ ID NO: 5378 0.52719 (genus)timonensis

TABLE 2 Deposited Microbes of the present disclosure Strain DesignationSEQ ID No: Deposit Accession # Ascusbbf_6176A 5379 PTA-125041,PTA-125042, PTA-125049, PTA-125050, PTA-125051, PTA-125052Ascusbbf_6176B 5380 PTA-125041, PTA-125042, PTA-125049, PTA-125050,PTA-125051, PTA-125052 Ascusbbf_6176C 5381 PTA-125041, PTA-125049,PTA-125050 Ascusbbf_6176D 5382 PTA-125049, PTA-125051, PTA-125052Ascusbbf_6176E 5383 PTA-125049, PTA-125051, PTA-125052 Ascusbbf_6176F5384 PTA-125049 Ascusbbf_6176G 5385 PTA-125049, PTA-125050, PTA-125051,PTA-125052 Ascusbbf_6176H 5386 PTA-125049, PTA-125051, PTA-125052Ascusbbf_6176I 5387 PTA-125050, PTA-125051, PTA-125052 Ascusbbf_4883A5388 PTA-125042, PTA-125049 Ascusbbf_4883B 5389 PTA-125049, PTA-125051Ascusbbf_4883C 5390 PTA-125049, PTA-125050, PTA-125051 Ascusbbf_4883D5391 PTA-125049, PTA-125050, PTA-125051 Ascusbbf_4883E 5392 PTA-125050Ascusbbf_13543A 5393 PTA-125033 Ascusbbf_13543B 5394 PTA-125041,PTA-125050 Ascusbbf_13543C 5395 PTA-125041, PTA-125042, PTA-125050,PTA-125051 Ascusbbf_13543D 5396 PTA-125041, PTA-125050 Ascusbbf_13543E5397 PTA-125033, PTA-125041, PTA-125042, PTA-125051, PTA-125052Ascusbbf_152A 5398 PTA-125051 Ascusbbf_152B 5399 PTA-125051Ascusbbf_152C 5400 PTA-125051 Ascusbbf_707A 5401 PTA-125049, PTA-125050,PTA-125051, PTA-125052 Ascusbbf_707B 5402 PTA-125049, PTA-125050,PTA-125051, PTA-125052 Ascusbbf_707C 5403 PTA-125049, PTA-125050Ascusbbf_707D 5404 PTA-125049, PTA-125051, PTA-125052 Ascusbbf_707E 5405PTA-125049, PTA-125051, PTA-125052 Ascusbbf_707F 5406 PTA-125049,PTA-125050, PTA-125051, PTA-125052 Ascusbbf_707G 5407 PTA-125049,PTA-125050, PTA-125051, PTA-125052 Ascusbbf_707H 5408 PTA-125049,PTA-125050, PTA-125051, PTA-125052 Ascusbbf_707I 5409 PTA-125051Ascusbbf_707J 5410 PTA-125051 Ascusbbf_1238A 5411 PTA-125033,PTA-125040, PTA-125041, PTA-125050, PTA-125051, PTA-125052Ascusbbf_1238B 5412 PTA-125040, PTA-125041, PTA-125050, PTA-125052Ascusbbf_1238C 5413 PTA-125033, PTA-125040, PTA-125041, PTA-125042,PTA-125050, PTA-125051, PTA-125052 Ascusbbf_1238D 5414 PTA-125033,PTA-125051, PTA-125052 Ascusbbf_5588A 5415 PTA-125040, PTA-125041,PTA-125042, PTA-125049, PTA-125050 Ascusbbf_5588B 5416 PTA-125040,PTA-125042 Ascusbbf_5588C 5417 PTA-125040, PTA-125041, PTA-125042,PTA-125049, PTA-125050, PTA-125051 Ascusbbf_5588D 5418 PTA-125040,PTA-125041, PTA-125042, PTA-125049, PTA-125050, PTA-125051, PTA-125052Ascusbbf_5588E 5419 PTA-125042 Ascusbbf_5588F 5420 PTA-125042Ascusbbf_5588G 5421 PTA-125042 Ascusbbf_5588H 5422 PTA-125049,PTA-125051 Ascusbbf_4691A 5423 PTA-125050, PTA-125051 Ascusbbf_4691B5424 PTA-125051, PTA-125052 Ascusbbf_4691C 5425 PTA-125051Ascusbbf_9770A 5426 PTA-125041, PTA-125042, PTA-125049, PTA-125050Ascusbbf_9770B 5427 PTA-125041, PTA-125049, PTA-125050 Ascusbbf_9770C5428 PTA-125049, PTA-125051, PTA-125052 Ascusbbf_9770D 5429 PTA-125049,PTA-125050 Ascusbbf_9770E 5430 PTA-125049, PTA-125050 Ascusbbf_9770F5431 PTA-125049, PTA-125050 Ascusbbf_9770G 5432 PTA-125049, PTA-125050Ascusbbf_9770H 5433 PTA-125049, PTA-125050, PTA-125051 Ascusbbf_14146A5434 PTA-124942, PTA-125033, PTA-125041, PTA-125042, PTA-125051,PTA-125052, B-67555 Ascusbbf_14146B 5435 PTA-125033, PTA-125041,PTA-125042, PTA-125051, PTA-125052 Ascusbbf_14146C 5436 PTA-125041Ascusbbf_14146D 5437 PTA-125033, PTA-125041, PTA-125042, PTA-125051,PTA-125052 Ascusbbf_14146E 5438 PTA-125033, PTA-125041 Ascusbbf_14146F5439 PTA-125041 Ascusbbf_14146G 5440 PTA-125042, PTA-125051, PTA-125052Ascusbbf_1103A 5441 PTA-125033, PTA-125041 Ascusbbf_1103B 5442PTA-125041, PTA-125051, PTA-125052 Ascusbbf_1103C 5443 PTA-125041,PTA-125051, PTA-125052 Ascusbbf_1103D 5444 PTA-125041, PTA-125042,PTA-125051 Ascusbbf_1103E 5445 PTA-125041, PTA-125051, PTA-125052Ascusbbf_1103F 5446 PTA-125051, PTA-125052 Ascusbbf_1103G 5447PTA-125051, PTA-125052 Ascusbbf_1103H 5448 PTA-125051, PTA-125052Ascusbbf_1103I 5449 PTA-125051 Ascusbbf_13717A 5450 PTA-125051,PTA-125052 Ascusbbf_13717B 5451 PTA-125051 Ascusbbf_13717C 5452PTA-125051 Ascusbbf_876A 5453 PTA-124942, PTA-125042 Ascusbbf_876B 5454PTA-125042 Ascusbbf_876C 5455 PTA-125033, PTA-125040, PTA-125041,PTA-125042, PTA-125050 Ascusbbf_876D 5456 PTA-125033, PTA-125040,PTA-125041, PTA-125042, PTA-125050, PTA-125052 Ascusbbf_876E 5457B-67553 Ascusbbf_876F 5458 PTA-125033, PTA-125040, PTA-125041,PTA-125042, PTA-125050, PTA-125052 Ascusbbf_876G 5459 PTA-125033,PTA-125042 Ascusbbf_4936A 5460 PTA-125041, PTA-125050 Ascusbbf_4936B5461 PTA-125041 Ascusbbf_113152A 5462 PTA-125050 Ascusbbf_9031A 5463PTA-125049 Ascusbbf_9031B 5464 PTA-125049 Ascusbbf_9031C 5465 PTA-125049Ascusbbf_9031D 5466 PTA-125049, PTA-125050 Ascusbbf_9031E 5467PTA-125049, PTA-125050 Ascusbbf_9031F 5468 PTA-125049, PTA-125050Ascusbbf_9031G 5469 PTA-125050 Ascusbbf_11823A 5470 PTA-125041Ascusbbf_11823B 5471 PTA-125041, PTA-125042, PTA-125049 Ascusbbf_11823C5472 PTA-125050 Ascusbbf_1007A 5473 PTA-125041 Ascusbbf_24422A 5474PTA-125051, PTA-125052 Ascusbbf_951A 5475 PTA-124942, PTA-125042Ascusbbf_951B 5476 PTA-125033, PTA-125040, PTA-125041, PTA-125042,PTA-125049, PTA-125050, PTA-125051, PTA-125052 Ascusbbf_951C 5477PTA-125033, PTA-125040, PTA-125041, PTA-125042, PTA-125049, PTA-125050,PTA-125051, PTA-125052 Ascusbbf_951D 5478 PTA-125033, PTA-125040,PTA-125041, PTA-125042, PTA-125049 Ascusbbf_951E 5479 PTA-125033,PTA-125042, PTA-125051, PTA-125052 Ascusbbf_951F 5480 PTA-125033Ascusbbf_951G 5481 PTA-125033 Ascusbbf_5699A 5482 PTA-125049, PTA-125050Ascusbbf_5699B 5483 PTA-125049, PTA-125050 Ascusbbf_5699C 5484PTA-125049, PTA-125050 Ascusbbf_5699D 5485 PTA-125050, PTA-125051Ascusbbf_130A 5486 PTA-125049, PTA-125050, PTA-125051, PTA-125052Ascusbbf_130B 5487 PTA-125049 Ascusbbf_130C 5488 PTA-125049, PTA-125050Ascusbbf_130D 5489 PTA-125049, PTA-125050 Ascusbbf_130E 5490 PTA-125049Ascusbbf_130F 5491 PTA-125049, PTA-125050 Ascusbbf_130G 5492 PTA-125051Ascusbbf_10109A 5493 PTA-124942 Ascusbbf_10109B 5494 PTA-124942Ascusbbf_10109C 5495 PTA-125033, PTA-125049 Ascusbbf_10109D 5496PTA-125049 Ascusbbf_10109E 5497 PTA-125049, PTA-125050 Ascusbbf_10109F5498 PTA-125049 Ascusbbf_10109G 5499 PTA-125049, PTA-125052Ascusbbf_10109H 5500 PTA-125049, PTA-125050 Ascusbbf_10109I 5501PTA-125050 Ascusbbf_29797A 5502 PTA-125051 Ascusbbf_54068A 5503PTA-124942 Ascusbbf_54068B 5504 PTA-125033 Ascusbbf_54068C 5505PTA-125033, PTA-125040, PTA-125041, PTA-125042, PTA-125049Ascusbbf_54068D 5506 PTA-125033, PTA-125041, PTA-125042, PTA-125049,PTA-125050 Ascusbbf_7003A 5507 PTA-124942 Ascusbbf_7003C 5508 PTA-125033Ascusbbf_23A 5509 PTA-125051, PTA-125052 Ascusbbf_1697A 5510 PTA-125049Ascusbbf_1697B 5511 PTA-125050 Ascusbbf_1697C 5512 PTA-125050Ascusbbf_7586A 5513 PTA-125041 Ascusbbf_7586B 5514 PTA-125041,PTA-125042, PTA-125050 Ascusbbf_7586C 5515 PTA-125041, PTA-125042,PTA-125050 Ascusbbf_7586D 5516 PTA-125042 Ascusbbf_1034A 5517PTA-125051, PTA-125052 Ascusbbf_1034B 5518 PTA-125051 Ascusbbf_23134A5519 PTA-125049 Ascusbbf_43679A 5520 PTA-125040, PTA-125041, PTA-125050Ascusbbf_43679B 5521 PTA-125041 Ascusbbf_43679C 5522 PTA-125041Ascusbbf_43679D 5523 PTA-125041 Ascusbbf_43679E 5524 PTA-125041,PTA-125050 Ascusbbf_43679F 5525 PTA-125050 Ascusbbf_63954A 5526PTA-125049 Ascusbbf_1517A 5527 PTA-125033, PTA-125040, PTA-125041,PTA-125042, PTA-125051, PTA-125052 Ascusbbf_1517B 5528 PTA-125040,PTA-125041, PTA-125042, PTA-125051, PTA-125052 Ascusbbf_1517C 5529PTA-125040, PTA-125042, PTA-125049, PTA-125050, PTA-125051Ascusbbf_1517D 5530 PTA-125033, PTA-125040, PTA-125042, PTA-125049,PTA-125050, PTA-125051, PTA-125052 Ascusbbf_1517E 5531 PTA-125033,PTA-125041, PTA-125050 Ascusbbf_1517F 5532 PTA-125042 Ascusbbf_1517G5533 PTA-125042 Ascusbbf_1517H 5534 PTA-125042 Ascusbbf_1517I 5535PTA-125049 Ascusbbf_104A 5536 PTA-124942 Ascusbbf_104B 5537 PTA-125040,PTA-125042, PTA-125049, PTA-125050 Ascusbbf_104C 5538 PTA-125040,PTA-125042, PTA-125049, PTA-125050 Ascusbbf_104D 5539 PTA-125040Ascusbbf_104E 5540 PTA-125040, PTA-125041, PTA-125042 Ascusbbf_104F 5541PTA-125033, PTA-125042 Ascusbbf_104G 5542 PTA-125050 Ascusbbf_104H 5543PTA-125042 Ascusbbf_104I 5544 PTA-125042 Ascusbbf_148A 5545 PTA-125049,PTA-125051, PTA-125052 Ascusbbf_148B 5546 PTA-125049, PTA-125051Ascusbbf_148C 5547 PTA-125049, PTA-125050, PTA-125051, PTA-125052Ascusbbf_148D 5548 PTA-125051, PTA-125052 Ascusbbf_148E 5549 PTA-125051,PTA-125052 Ascusbbf_148F 5550 PTA-125051, PTA-125052 Ascusbbf_148G 5551PTA-125051 Ascusbbf_148H 5552 PTA-125051 Ascusbbf_944A 5553 PTA-124942Ascusbbf_944B 5554 PTA-125041, PTA-125049, PTA-125051, PTA-125052Ascusbbf_944C 5555 PTA-125049, PTA-125051 Ascusbbf_944D 5556 PTA-125049,PTA-125050 Ascusbbf_944E 5557 PTA-125049 Ascusbbf_944F 5558 PTA-125049Ascusbbf_944G 5559 PTA-125049, PTA-125051, PTA-125052 Ascusbbf_23033A5560 PTA-125051, PTA-125052 Ascusbbf_23033B 5561 PTA-125051, PTA-125052Ascusbbf_23033C 5562 PTA-125051, PTA-125052 Ascusbbf_23033D 5563PTA-125051 Ascusbbf_2600A 5564 PTA-124942 Ascusbbf_2600B 5565PTA-125033, PTA-125040, PTA-125041, PTA-125042 Ascusbbf_2600C 5566PTA-125033, PTA-125040, PTA-125041, PTA-125042 Ascusbbf_2600D 5567PTA-125041 Ascusbbf_2600E 5568 PTA-125033, PTA-125041 Ascusbbf_2600F5569 PTA-125033, PTA-125041 Ascusbbf_2600G 5570 PTA-125041Ascusbbf_2600H 5571 PTA-125042 Ascusbbf_8118A 5572 PTA-125051,PTA-125052 Ascusbbf_8118B 5573 PTA-125051 Ascusbbf_201A 5574 PTA-125042Ascusbbf_201J 5575 PTA-125033, PTA-125042, PTA-125049 Ascusbbf_201K 5576PTA-125042, PTA-125049, PTA-125051, PTA-125052 Ascusbbf_201L 5577PTA-125042, PTA-125052 Ascusbbf_10712A 5578 PTA-124942, PTA-125033,PTA-125042, PTA-125050 Ascusbbf_10712B 5579 PTA-124942 Ascusbbf_10712C5580 PTA-125033, PTA-125042, PTA-125049 Ascusbbf_10712D 5581 PTA-125033,PTA-125042 Ascusbbf_10712E 5582 PTA-125042, PTA-125049 Ascusbbf_10712F5583 PTA-125033, PTA-125042, PTA-125050 Ascusbbf_10712G 5584 PTA-125033,PTA-125042, PTA-125049, PTA-125050, PTA-125052 Ascusbbf_10712H 5585PTA-125033, PTA-125042, PTA-125049, PTA-125050, PTA-125052Ascusbbf_10712I 5586 PTA-125042, PTA-125050 Ascusbbf_6012A 5587PTA-124942 Ascusbbf_6012B 5588 PTA-125040, PTA-125041, PTA-125049,PTA-125050 Ascusbbf_6012C 5589 PTA-125040, PTA-125041, PTA-125049,PTA-125050 Ascusbbf_6012D 5590 PTA-125041, PTA-125049 Ascusbbf_6012E5591 PTA-125041 Ascusbbf_2297A 5592 PTA-125049, PTA-125050, PTA-125051Ascusbbf_2297B 5593 PTA-125049, PTA-125051 Ascusbbf_2297C 5594PTA-125049, PTA-125051, PTA-125052 Ascusbbf_2297D 5595 PTA-125049,PTA-125051, PTA-125052 Ascusbbf_2297E 5596 PTA-125049, PTA-125051,PTA-125052 Ascusbbf_2297F 5597 PTA-125051, PTA-125052 Ascusbbf_2297G5598 PTA-125051, PTA-125052 Ascusbbf_2297H 5599 PTA-125051 Ascusbbf_873A5600 PTA-125033 Ascusbbf_873B 5601 PTA-125033, PTA-125041, PTA-125042,PTA-125049, PTA-125050, PTA-125052 Ascusbbf_873C 5602 PTA-125040,PTA-125041, PTA-125042, PTA-125050 Ascusbbf_873D 5603 PTA-125041,PTA-125042, PTA-125050 Ascusbbf_873E 5604 PTA-125033, PTA-125041,PTA-125042, PTA-125049, PTA-125050 Ascusbbf_873F 5605 PTA-125033,PTA-125041, PTA-125042, PTA-125052 Ascusbbf_873G 5606 PTA-125042Ascusbbf_269A 5607 PTA-125033, PTA-125041, PTA-125042, PTA-125050Ascusbbf_269B 5608 PTA-125033 Ascusbbf_269C 5609 PTA-125033, PTA-125042Ascusbbf_269D 5610 PTA-125041, PTA-125050 Ascusbbf_269E 5611 PTA-125033,PTA-125041, PTA-125050 Ascusbbf_269F 5612 PTA-125033 Ascusbbf_269G 5613PTA-125033 Ascusbbf_41015A 5614 PTA-125033, PTA-125041, PTA-125050Ascusbbf_41015B 5615 PTA-125033 Ascusbbf_41015C 5616 PTA-125041Ascusbbf_41015D 5617 PTA-125042 Ascusbbf_41015E 5618 PTA-125042Ascusbbf_32877A 5619 PTA-124942 Ascusbbf_57294A 5620 PTA-124942,PTA-125041 Ascusbbf_57294B 5621 PTA-125033, PTA-125050 Ascusbbf_57294C5622 PTA-125033, PTA-125052 Ascusbbf_27932A 5623 PTA-125040Ascusbbf_27932B 5624 PTA-125040, PTA-125041, PTA-125050 Ascusbbf_27932C5625 PTA-125040, PTA-125049, PTA-125052 Ascusbbf_22558A 5626 PTA-125051Ascusbbf_22558B 5627 PTA-125051 Ascusbbf_983757A 5628 PTA-125051,PTA-125052 Ascusbbf_983757B 5629 PTA-125051, PTA-125052 Ascusbbf_983757C5630 PTA-125051, PTA-125052 Ascusbbf_52330A 5631 PTA-125033Ascusbbf_1085A 5632 PTA-124942, PTA-125033, PTA-125041, PTA-125049,PTA-125050 Ascusbbf_1085B 5633 PTA-125033, PTA-125042, PTA-125050,PTA-125051, B-67554 Ascusbbf_1085C 5634 PTA-125033, PTA-125040,PTA-125041, PTA-125042, PTA-125049, PTA-125050, PTA-125051, PTA-125052Ascusbbf_1085D 5635 PTA-125040, PTA-125041, PTA-125042, PTA-125049,PTA-125050, PTA-125052 Ascusbbf_1085E 5636 PTA-125033, PTA-125040,PTA-125041, PTA-125042, PTA-125049, PTA-125050, PTA-125052Ascusbbf_1085F 5637 PTA-125033, PTA-125049, PTA-125051, PTA-125052Ascusbbf_1085G 5638 PTA-125033, PTA-125050 Ascusbbf_1085H 5639PTA-125033, PTA-125050 Ascusbbf_1085I 5640 PTA-125033, PTA-125050Ascusbbf_1085J 5641 PTA-125033, PTA-125042 Ascusbbf_1085K 5642PTA-125042 Ascusbbf_154A 5643 PTA-124942 Ascusbbf_154B 5644 PTA-125042,PTA-125049, PTA-125050, PTA-125051, B-67550 Ascusbbf_154D 5645PTA-125040, PTA-125041, PTA-125042, PTA-125051 Ascusbbf_154E 5646PTA-125040, PTA-125041, PTA-125049, PTA-125050, PTA-125052 Ascusbbf_154F5647 PTA-125033, PTA-125040, PTA-125041, PTA-125042, PTA-125049,PTA-125050, PTA-125052 Ascusbbf_154G 5648 PTA-125033, PTA-125041,PTA-125042, PTA-125049, PTA-125050, PTA-125051, PTA-125052 Ascusbbf_154H5649 PTA-125049, PTA-125050 Ascusbbf_154I 5650 PTA-125049, PTA-125050Ascusbbf_154M 5651 PTA-125042 Ascusbbf_1010A 5652 PTA-124942Ascusbbf_1010B 5653 PTA-125033, PTA-125040, PTA-125041, PTA-125042,PTA-125049, PTA-125050, PTA-125051, PTA-125052 Ascusbbf_1010C 5654PTA-125040, PTA-125041, PTA-125042, PTA-125049, PTA-125050, PTA-125051,PTA-125052 Ascusbbf_1010D 5655 PTA-125033, PTA-125040, PTA-125041,PTA-125042, PTA-125049, PTA-125050, PTA-125051, PTA-125052Ascusbbf_1010E 5656 PTA-125033, PTA-125040, PTA-125041, PTA-125042,PTA-125052 Ascusbbf_1010F 5657 PTA-125033, PTA-125040, PTA-125041,PTA-125052 Ascusbbf_1010G 5658 PTA-125033, PTA-125040, PTA-125041,PTA-125042, PTA-125049, PTA-125050, PTA-125051, PTA-125052Ascusbbf_1010H 5659 PTA-125033, PTA-125041 Ascusbbf_1010I 5660PTA-125033, PTA-125041, PTA-125051 Ascusbbf_1010J 5661 PTA-125041,PTA-125050, PTA-125052 Ascusbbf_5575A 5662 PTA-124942 Ascusbbf_5575B5663 PTA-124942 Ascusbbf_5575C 5664 PTA-125033, PTA-125042, PTA-125050Ascusbbf_5575D 5665 PTA-125042, PTA-125050 Ascusbbf_5575E 5666PTA-125033, PTA-125042, PTA-125049, PTA-125052 Ascusbbf_5575F 5667PTA-125033, PTA-125042, PTA-125049 Ascusbbf_5575G 5668 PTA-125033,PTA-125042, PTA-125049, PTA-125050 Ascusbbf_5575H 5669 PTA-125033,PTA-125042, PTA-125050 Ascusbbf_775A 5670 PTA-125049, PTA-125050,PTA-125051, PTA-125052 Ascusbbf_775B 5671 PTA-125051 Ascusbbf_24302A5672 PTA-125041, PTA-125050, B-67551 Ascusbbf_24302B 5673 PTA-125033,PTA-125040, PTA-125041, PTA-125049 Ascusbbf_24302C 5674 PTA-125033,PTA-125040, PTA-125041, PTA-125051, PTA-125052 Ascusbbf_24302D 5675PTA-125041, PTA-125049 Ascusbbf_24302E 5676 PTA-125033, PTA-125041,PTA-125052 Ascusbbf_24302F 5677 PTA-125033, PTA-125041, PTA-125042,PTA-125049, PTA-125050, PTA-125052 Ascusbbf_24302G 5678 PTA-125033,PTA-125041, PTA-125049, PTA-125050, PTA-125051, PTA-125052Ascusbbf_24302H 5679 PTA-125041, PTA-125052 Ascusbbf_24302I 5680PTA-125041, PTA-125050, PTA-125051, PTA-125052 Ascusbbf_24302J 5681PTA-125051, PTA-125052 Ascusbbf_1A 5682 PTA-125040, PTA-125041,PTA-125042, PTA-125052 Ascusbbf_1B 5683 PTA-125040, PTA-125041,PTA-125042, PTA-125049, PTA-125050, PTA-125052 Ascusbbf_1C 5684PTA-125040, PTA-125041, PTA-125042, PTA-125050 Ascusbbf_1D 5685PTA-125033, PTA-125040, PTA-125041, PTA-125042, PTA-125049, PTA-125050,PTA-125051, PTA-125052 Ascusbbf_1E 5686 PTA-125033, PTA-125040,PTA-125042 Ascusbbf_1F 5687 PTA-125033, PTA-125040, PTA-125041,PTA-125042, PTA-125050, PTA-125051, PTA-125052 Ascusbbf_1G 5688PTA-125033, PTA-125040, PTA-125041, PTA-125042, PTA-125049, PTA-125050,PTA-125052 Ascusbbf_1H 5689 PTA-125033, PTA-125040, PTA-125041,PTA-125050 Ascusbbf_1I 5690 PTA-125033, PTA-125040, PTA-125041,PTA-125042, PTA-125050, PTA-125052 Ascusbbf_1J 5691 PTA-125033,PTA-125040, PTA-125041, PTA-125042, PTA-125049, PTA-125050 Ascusbbf_1K5692 PTA-125041, PTA-125042, PTA-125049, PTA-125050 Ascusbbf_52548A 5693PTA-125051, PTA-125052 Ascusbbf_52548B 5694 PTA-125051, PTA-125052Ascusbbf_50658A 5695 PTA-125050 Ascusbbf_850A 5696 PTA-124942Ascusbbf_850B 5697 PTA-125033 Ascusbbf_850C 5698 PTA-125033, PTA-125040,PTA-125041, PTA-125049, PTA-125050 Ascusbbf_850D 5699 PTA-125040Ascusbbf_850E 5700 PTA-125040, PTA-125041, PTA-125042, PTA-125049,PTA-125050 Ascusbbf_850F 5701 PTA-125033, PTA-125040, PTA-125041,PTA-125042, PTA-125049, PTA-125050 Ascusbbf_850G 5702 PTA-125033,PTA-125040, PTA-125041, PTA-125049, PTA-125050 Ascusbbf_850H 5703PTA-125033, PTA-125041, PTA-125050 Ascusbbf_850I 5704 PTA-125033,PTA-125050 Ascusbbf_850J 5705 PTA-125033, PTA-125042 Ascusbbf_4A 5706PTA-124942 Ascusbbf_4B 5707 PTA-125040, PTA-125041, PTA-125042,PTA-125049, PTA-125050, PTA-125052 Ascusbbf_4C 5708 PTA-125041,PTA-125051 Ascusbbf_4D 5709 PTA-125040, PTA-125041, PTA-125042,PTA-125050, PTA-125051, PTA-125052, B-67552 Ascusbbf_4E 5710 PTA-125040,PTA-125041, PTA-125050, PTA-125052 Ascusbbf_4F 5711 PTA-125033,PTA-125040, PTA-125041, PTA-125042, PTA-125050, PTA-125051, PTA-125052Ascusbbf_4G 5712 PTA-125041, PTA-125042, PTA-125049, PTA-125050,PTA-125051, PTA-125052 Ascusbbf_4H 5713 PTA-125041, PTA-125042,PTA-125049, PTA-125050, PTA-125051, PTA-125052 Ascusbbf_4I 5714PTA-125041, PTA-125049, PTA-125050, PTA-125052 Ascusbbf_4J 5715PTA-125041, PTA-125050 Ascusbbf_4K 5716 PTA-125051 Ascusbbf_5131A 5717PTA-125049, PTA-125050, PTA-125051 Ascusbbf_5131B 5718 PTA-125049,PTA-125051 Ascusbbf_5131C 5719 PTA-125049, PTA-125051 Ascusbbf_5131D5720 PTA-125049, PTA-125051 Ascusbbf_5131E 5721 PTA-125051, PTA-125052Ascusbbf_5131F 5722 PTA-125051 Ascusbbf_5131G 5723 PTA-125051Ascusbbf_5131H 5724 PTA-125051 Ascusbbf_8600A 5725 PTA-125051Ascusbbf_8600B 5726 PTA-125051 Ascusbbf_1273A 5727 PTA-125049,PTA-125051, PTA-125052 Ascusbbf_1273B 5728 PTA-125051 Ascusbbf_1273C5729 PTA-125051 Ascusbbf_1273D 5730 PTA-125051 Ascusbbf_39159A 5731PTA-125041 Ascusbbf_39159B 5732 PTA-125041 Ascusbbf_39159C 5733PTA-125050 Ascusbbf_39159D 5734 PTA-125042 Ascusbbf_318A 5735PTA-125049, PTA-125050 Ascusbbf_318B 5736 PTA-125049 Ascusbbf_318C 5737PTA-125049, PTA-125050 Ascusbbf_318D 5738 PTA-125042, PTA-125050,PTA-125051 Ascusbbf_318E 5739 PTA-125042, PTA-125050, PTA-125051,PTA-125052 Ascusbbf_7046A 5740 PTA-125050 Ascusbbf_1372985A 5741PTA-124942, PTA-125041 Ascusbbf_1372985B 5742 PTA-125033, PTA-125041,PTA-125042 Ascusbbf_1372985C 5743 PTA-125033, PTA-125041Ascusbbf_1372985D 5744 PTA-125040, PTA-125041, PTA-125042, PTA-125049,PTA-125050 Ascusbbf_1372985E 5745 PTA-125040, PTA-125041, PTA-125049,PTA-125050 Ascusbbf_1372985F 5746 PTA-125041 Ascusbbf_1372985G 5980PTA-125033, PTA-125041 Ascusbbf_1372985H 5981 PTA-125033, PTA-125041Ascusbbf_1372985I 5747 PTA-125033, PTA-125041 Ascusbbf_1372985J 5982PTA-125033, PTA-125041, PTA-125042, PTA-125051, PTA-125052Ascusbbf_1372985K 5983 PTA-125033, PTA-125041, PTA-125050, PTA-125051Ascusbbf_1372985L 5748 PTA-125041 Ascusbbf_1372985M 5749 PTA-125041Ascusbbf_1372985N 5750 PTA-125041 Ascusbbf_1372985O 5751 PTA-125041Ascusbbf_1372985P 5752 PTA-125041 Ascusbbf_1372985Q 5753 PTA-125041Ascusbbf_1372985R 5754 PTA-125041 Ascusbbf_1372985S 5755 PTA-125041,PTA-125049, PTA-125050 Ascusbbf_1372985T 5756 PTA-125041, PTA-125049,PTA-125050 Ascusbbf_121971A 5757 PTA-125041 Ascusbbf_5251A 5758PTA-125033, PTA-125049, PTA-125050 Ascusbbf_5251B 5759 PTA-125033,PTA-125041 Ascusbbf_5251C 5760 PTA-125041, PTA-125049, PTA-125050,PTA-125051 Ascusbbf_5251D 5761 PTA-125033, PTA-125041, PTA-125049Ascusbbf_5251E 5762 PTA-125049, PTA-125050, PTA-125051 Ascusbbf_5251F5763 PTA-125051, PTA-125052 Ascusbbf_5251G 5764 PTA-125051 Ascusbbf_100A5765 PTA-125033, PTA-125049, PTA-125050, PTA-125052 Ascusbbf_100B 5766PTA-125040, PTA-125042 Ascusbbf_100C 5767 PTA-125033, PTA-125040,PTA-125041, PTA-125042, PTA-125049, PTA-125050, PTA-125052 Ascusbbf_100D5768 PTA-125033, PTA-125040, PTA-125041, PTA-125050 Ascusbbf_100E 5769PTA-125033, PTA-125040, PTA-125041, PTA-125042, PTA-125049, PTA-125050Ascusbbf_100F 5770 PTA-125041 Ascusbbf_100G 5771 PTA-125042Ascusbbf_20584A 5772 PTA-125049 Ascusbbf_20584B 5773 PTA-125051,PTA-125052 Ascusbbf_4317A 5774 PTA-125041 Ascusbbf_4317B 5775 PTA-125041Ascusbbf_4317C 5776 PTA-125042 Ascusbbf_4317D 5777 PTA-125051,PTA-125052 Ascusbbf_4317E 5778 PTA-125051, PTA-125052 Ascusbbf_6A 5779PTA-125040, PTA-125041, PTA-125049, PTA-125050, PTA-125052 Ascusbbf_6B5780 PTA-125040, PTA-125041, PTA-125042, PTA-125049, PTA-125050,PTA-125052 Ascusbbf_6C 5781 PTA-125040, PTA-125041, PTA-125042,PTA-125049, PTA-125050, PTA-125052 Ascusbbf_6D 5782 PTA-125040,PTA-125041, PTA-125050, PTA-125052 Ascusbbf_6E 5783 PTA-125040,PTA-125041, PTA-125042, PTA-125049, PTA-125050, PTA-125052 Ascusbbf_6F5784 PTA-125040, PTA-125052 Ascusbbf_6G 5785 PTA-125040, PTA-125041,PTA-125042, PTA-125049, PTA-125050, PTA-125052 Ascusbbf_6H 5786PTA-125041, PTA-125042, PTA-125050 Ascusbbf_6I 5787 PTA-125041,PTA-125050 Ascusbbf_6J 5788 PTA-125041, PTA-125050 Ascusbbf_6K 5789PTA-125042 Ascusbbf_6L 5790 PTA-125042 Ascusbbf_19022A 5791 PTA-125033,PTA-125040, PTA-125041 Ascusbbf_19022B 5792 PTA-125040, PTA-125041,PTA-125042, PTA-125050 Ascusbbf_19022C 5793 PTA-125042, PTA-125052Ascusbbf_2624A 5794 PTA-125033, PTA-125041, PTA-125042, PTA-125051,PTA-125052 Ascusbbf_2624B 5795 PTA-125041, PTA-125042, PTA-125050,PTA-125051, PTA-125052 Ascusbbf_2624C 5796 PTA-125042, PTA-125051,PTA-125052 Ascusbbf_2624D 5797 PTA-125042, PTA-125051, PTA-125052Ascusbbf_2624E 5798 PTA-125042, PTA-125051, PTA-125052 Ascusbbf_2624F5799 PTA-125051, PTA-125052 Ascusbbf_2624G 5800 PTA-125051Ascusbbf_3427A 5801 PTA-125041, PTA-125049, PTA-125050 Ascusbbf_3427B5802 PTA-125041, PTA-125042, PTA-125049, PTA-125050, PTA-125051,PTA-125052 Ascusbbf_3427C 5803 PTA-125042, PTA-125049, PTA-125050,PTA-125051 Ascusbbf_3427D 5804 PTA-125050 Ascusbbf_3427E 5805 PTA-125052Ascusbbf_5005A 5806 PTA-125049, PTA-125050, PTA-125051 Ascusbbf_5005B5807 PTA-125050 Ascusbbf_69A 5808 PTA-125033, PTA-125040, PTA-125041,PTA-125042, PTA-125049, PTA-125050, PTA-125051, PTA-125052 Ascusbbf_69B5809 PTA-125033, PTA-125040, PTA-125041, PTA-125042, PTA-125049,PTA-125050, PTA-125051, PTA-125052 Ascusbbf_69C 5810 PTA-125033,PTA-125040, PTA-125041, PTA-125042, PTA-125049, PTA-125050, PTA-125051,PTA-125052 Ascusbbf_69D 5811 PTA-125033, PTA-125040, PTA-125041,PTA-125049, PTA-125050, PTA-125051, PTA-125052 Ascusbbf_69E 5812PTA-125033, PTA-125040, PTA-125041, PTA-125042, PTA-125049, PTA-125050,PTA-125051, PTA-125052 Ascusbbf_69F 5813 PTA-125033, PTA-125040,PTA-125041, PTA-125042, PTA-125049, PTA-125050, PTA-125051, PTA-125052Ascusbbf_69G 5814 PTA 125033, PTA-125041, PTA-125042, PTA-125049,PTA-125050, PTA-125051, PTA-125052 Ascusbbf_69H 5815 PTA-125033,PTA-125041, PTA-125042, PTA-125049, PTA-125050, PTA-125051, PTA-125052Ascusbbf_69I 5816 PTA-125033, PTA-125041, PTA-125042, PTA-125049,PTA-125051, PTA-125052 Ascusbbf_69J 5817 PTA-125033, PTA-125042,PTA-125051, PTA-125052 Ascusbbf_8082A 5818 PTA-125033, PTA-125040,PTA-125041, PTA-125042, PTA-125050, PTA-125052 Ascusbbf_8082B 5819PTA-125040, PTA-125041, PTA-125042, PTA-125050 Ascusbbf_8082C 5820PTA-125033, PTA-125040, PTA-125041, PTA-125042, PTA-125050, PTA-125052Ascusbbf_8082D 5821 PTA-125033, PTA-125040, PTA-125041, PTA-125042,PTA-125050, PTA-125052 Ascusbbf_8082E 5822 PTA-125033, PTA-125041Ascusbbf_8082F 5823 PTA-125033, PTA-125041, PTA-125042 Ascusbbf_8082G5824 PTA-125033, PTA-125042 Ascusbbf_8082H 5825 PTA-125033, PTA-125042Ascusbbf_8082I 5826 PTA-125033 Ascusbbf_95A 5827 PTA-125049, PTA-125050Ascusbbf_95B 5828 PTA-125049, PTA-125050, PTA-125051, PTA-125052Ascusbbf_95C 5829 PTA-125049, PTA-125050 Ascusbbf_95D 5830 PTA-125049,PTA-125050, PTA-125051, PTA-125052 Ascusbbf_95E 5831 PTA-125051,PTA-125052 Ascusbbf_95F 5832 PTA-125051, PTA-125052 Ascusbbf_95G 5833PTA-125051, PTA-125052 Ascusbbf_95H 5834 PTA-125051 Ascusbbf_1136A 5835PTA-125040, PTA-125041, PTA-125042, PTA-125049, PTA-125050Ascusbbf_1136B 5836 PTA-125040, PTA-125041, PTA-125042, PTA-125049,PTA-125050, PTA-125052 Ascusbbf_1136C 5837 PTA-125033, PTA-125040,PTA-125041, PTA-125042, PTA-125049, PTA-125050 Ascusbbf_1136D 5838PTA-125040, PTA-125041, PTA-125042, PTA-125049, PTA-125050, PTA-125052Ascusbbf_1136E 5839 PTA-125040, PTA-125041, PTA-125049, PTA-125050Ascusbbf_1136F 5840 PTA-125042 Ascusbbf_2770A 5841 PTA-125042,PTA-125049, PTA-125050, PTA-125051 Ascusbbf_2770B 5842 PTA-125049,PTA-125050 Ascusbbf_2770C 5843 PTA-125049 Ascusbbf_1629A 5844 PTA-125033Ascusbbf_1629B 5845 PTA-125040, PTA-125041 Ascusbbf_1629C 5846PTA-125040, PTA-125041, PTA-125042, PTA-125049, PTA-125050Ascusbbf_1629D 5847 PTA-125033, PTA-125040, PTA-125041, PTA-125049,PTA-125050 Ascusbbf_1629E 5848 PTA-125041, PTA-125050 Ascusbbf_1629F5849 PTA-125050 Ascusbbf_1629G 5850 PTA-125050 Ascusbbf_1821A 5851PTA-125049 Ascusbbf_1821B 5852 PTA-125049, PTA-125050 Ascusbbf_1821C5853 PTA-125049, PTA-125050 Ascusbbf_1821D 5854 PTA-125049, PTA-125050Ascusbbf_56782A 5855 PTA-125033, PTA-125041, PTA-125042, PTA-125049,PTA-125050, PTA-125051 Ascusbbf_56782B 5856 PTA-125051 Ascusbbf_56782C5857 PTA-125051 Ascusbbf_92A 5858 PTA-125040, PTA-125050 Ascusbbf_92B5859 PTA-125040, PTA-125041, PTA-125049, PTA-125050 Ascusbbf_92C 5860PTA-125040, PTA-125050 Ascusbbf_92D 5861 PTA-125040, PTA-125049,PTA-125050, PTA-125052 Ascusbbf_92E 5862 PTA-125040 Ascusbbf_92F 5863PTA-125040, PTA-125049, PTA-125050 Ascusbbf_92G 5984 PTA-125041,PTA-125042 Ascusbbf_92H 5864 PTA-125041, PTA-125049 Ascusbbf_92I 5985PTA-125041 Ascusbbf_92J 5986 PTA-125041 Ascusbbf_92K 5865 PTA-125041,PTA-125051, PTA-125052 Ascusbbf_92L 5866 PTA-125050 Ascusbbf_118A 5867PTA-125049, PTA-125051 Ascusbbf_118B 5868 PTA-125051, PTA-125052Ascusbbf_118C 5869 PTA-125051 Ascusbbf_5429A 5870 PTA-125040,PTA-125041, PTA-125049, PTA-125050, PTA-125052 Ascusbbf_5429B 5871PTA-125040, PTA-125041, PTA-125049, PTA-125050, PTA-125052Ascusbbf_5429C 5872 PTA-125033, PTA-125042 Ascusbbf_5429D 5873PTA-125049 Ascusbbf_3A 5874 PTA-125033, PTA-125040, PTA-125041,PTA-125049, PTA-125050, PTA-125052 Ascusbbf_3B 5875 PTA-125040,PTA-125041, PTA-125049, PTA-125050, PTA-125052 Ascusbbf_3C 5876PTA-125040, PTA-125041, PTA-125049, PTA-125050 Ascusbbf_3D 5877PTA-125040 Ascusbbf_3E 5878 PTA-125042 Ascusbbf_3F 5879 PTA-125042Ascusbbf_3G 5880 PTA-125050 Ascusbbf_10576A 5881 PTA-125049, PTA-125050,PTA-125051 Ascusbbf_10576B 5882 PTA-125049, PTA-125050, PTA-125051,PTA-125052 Ascusbbf_10576C 5883 PTA-125049, PTA-125050, PTA-125051Ascusbbf_10576D 5884 PTA-125049 Ascusbbf_10576E 5885 PTA-125051Ascusbbf_729A 5886 PTA-125049, PTA-125051 Ascusbbf_729B 5887 PTA-125051,PTA-125052 Ascusbbf_729C 5888 PTA-125051 Ascusbbf_729D 5889 PTA-125051Ascusbbf_729E 5890 PTA-125051 Ascusbbf_729F 5891 PTA-125051Ascusbbf_201A 5892 PTA-125042 Ascusbbf_201B 5893 PTA-125041, PTA-125042,PTA-125050, PTA-125051, PTA-125052 Ascusbbf_201C 5894 PTA-125033Ascusbbf_201D 5895 PTA-125033, PTA-125041, PTA-125042, PTA-125051,PTA-125052 Ascusbbf_201E 5896 PTA-125040, PTA-125041 Ascusbbf_201F 5897PTA-125040 Ascusbbf_201G 5898 PTA-125033, PTA-125041, PTA-125050Ascusbbf_201H 5899 PTA-125033, PTA-125041, PTA-125042, PTA-125050Ascusbbf_201I 5900 PTA-125033, PTA-125041, PTA-125042, PTA-125050,PTA-125051, PTA-125052 Ascusbbf_416A 5901 PTA-125049 Ascusbbf_416B 5902PTA-125049, PTA-125051, PTA-125052 Ascusbbf_416C 5903 PTA-125049,PTA-125050 Ascusbbf_416D 5904 PTA-125049, PTA-125050 Ascusbbf_416E 5905PTA-125049 Ascusbbf_416F 5906 PTA-125049, PTA-125051 Ascusbbf_416G 5907PTA-125050 Ascusbbf_15806A 5908 PTA-125049 Ascusbbf_15806B 5909PTA-125049, PTA-125051, PTA-125052 Ascusbbf_6115A 5910 PTA-125033,PTA-125041 Ascusbbf_6115B 5911 PTA-125041, PTA-125050 Ascusbbf_6115C5912 PTA-125041, PTA-125050 Ascusbbf_6115D 5913 PTA-125041Ascusbbf_1325058A 5914 PTA-125041, PTA-125049, PTA-125051Ascusbbf_1325058B 5915 PTA-125041, PTA-125042, PTA-125049, PTA-125050,PTA-125051 Ascusbbf_1325058C 5916 PTA-125049 Ascusbbf_1325058D 5917PTA-125049 Ascusbbf_1325058E 5918 PTA-125050 Ascusbbf_1325058F 5919PTA-125051 Ascusbbf_28350A 5920 PTA-125041, PTA-125050 Ascusbbf_28350B5921 PTA-125041, PTA-125050 Ascusbbf_28350C 5922 PTA-125041, PTA-125050Ascusbbf_372A 5923 PTA-125033, PTA-125040, PTA-125041, PTA-125042,PTA-125049, PTA-125050, PTA-125052 Ascusbbf_372B 5924 PTA-125033Ascusbbf_372C 5925 PTA-125040, PTA-125041, PTA-125049, PTA-125050Ascusbbf_372D 5926 PTA-125033, PTA-125040, PTA-125041 Ascusbbf_372E 5927PTA-125033, PTA-125040, PTA-125041, PTA-125042, PTA-125049, PTA-125050Ascusbbf_372F 5928 PTA-125033, PTA-125041, PTA-125050 Ascusbbf_372G 5929PTA-125033, PTA-125042 Ascusbbf_667A 5930 PTA-125042, PTA-125049,PTA-125050, PTA-125052 Ascusbbf_667B 5931 PTA-125042, PTA-125049Ascusbbf_1207A 5932 PTA-125033, PTA-125042, PTA-125049, PTA-125050Ascusbbf_1207B 5933 PTA-125033, PTA-125042 Ascusbbf_1207C 5934PTA-125033 Ascusbbf_1207D 5935 PTA-125040, PTA-125041 Ascusbbf_1207E5936 PTA-125040, PTA-125041 Ascusbbf_1207F 5937 PTA-125040, PTA-125041,PTA-125042 Ascusbbf_1207G 5938 PTA-125033, PTA-125040, PTA-125041,PTA-125042, PTA-125049, PTA-125050, PTA-125052 Ascusbbf_1207H 5939PTA-125033, PTA-125040 Ascusbbf_1207I 5940 PTA-125033, PTA-125040,PTA-125041, PTA-125049, PTA-125050 Ascusbbf_1207J 5941 PTA-125033,PTA-125042, PTA-125049 Ascusbbf_3875A 5942 PTA-125033, PTA-125041,PTA-125042 Ascusbbf_3875B 5943 PTA-125040, PTA-125042 Ascusbbf_3875C5944 PTA-125033, PTA-125041 Ascusbbf_3875D 5945 PTA-125041Ascusbbf_72889A 5946 PTA-125051, PTA-125052 Ascusbbf_72889B 5947PTA-125051, PTA-125052 Ascusbbf_106863A 5948 PTA-125051, PTA-125052Ascusbbf_106863B 5949 PTA-125051 Ascusbbf_120A 5987 PTA-125051Ascusbbf_120B 5950 PTA-125051 Ascusbbf_120C 5951 PTA-125051Ascusbbf_1207K 5952 PTA-125033, PTA-125049, PTA-125050 Ascusbbf_1207L5953 PTA-125049 Ascusbbf_930A 5954 PTA-125051, PTA-125052 Ascusbbf_930B5988 PTA-125051 Ascusbbf_915A 5955 PTA-125051 Ascusbbf_8941A 5956PTA-125051, PTA-125052 Ascusbbf_8480A 5989 PTA-125051, PTA-125052Ascusbbf_8480B 5957 PTA-125051 Ascusbbf_374A 5990 PTA-125051, PTA-125052Ascusbbf_374B 5958 PTA-125051, PTA-125052 Ascusbbf_374C 5991 PTA-125051,PTA-125052 Ascusbbf_6906A 5959 PTA-125051, PTA-125052 Ascusbbf_6906B5960 PTA-125051, PTA-125052 Ascusbbf_6906C 5992 PTA-125051, PTA-125052Ascusbbf_6906D 5961 PTA-125051 Ascusbbf_6906E 5962 PTA-125051Ascusbbf_69K 5963 PTA-125033, PTA-125042 Ascusbbf_69L 5993 PTA-125033,PTA-125042 Ascusbbf_69M 5964 PTA-125033, PTA-125042 Ascusbbf_69N 5965PTA-125033, PTA-125042 Ascusbbf_69O 5966 PTA-125033, PTA-125042Ascusbbf_69P 5967 PTA-125033 Ascusbbf_721A 5968 PTA-125051, PTA-125052Ascusbbf_721B 5969 PTA-125051, PTA-125052 Ascusbbf_721C 5970 PTA-125051,PTA-125052 Ascusbbf_3819A 5971 PTA-125051 Ascusbbf_4323A 5972PTA-125051, PTA-125052 Ascusbbf_4323B 5973 PTA-125051, PTA-125052Ascusbbf_4323C 5974 PTA-125051, PTA-125052 Ascusbbf_4323D 5975PTA-125051 Ascusbbf_6087A 5976 PTA-125051, PTA-125052 Ascusbbf_6087B5977 PTA-125051, PTA-125052 Ascusbbf_6087C 5978 PTA-125051Ascusbbf_8414A 5979 PTA-125051

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 shows a general workflow of one embodiment of the method fordetermining the absolute abundance of one or more active microorganismstrains.

FIG. 2 shows a general workflow of one embodiment of a method fordetermining the co-occurrence of one or more, or two or more, activemicroorganism strains in a sample with one or more metadata(environmental) parameters, followed by leveraging cluster analysis andcommunity detection methods on the network of determined relationships.

FIG. 3 depicts a diagram that exemplifies how the diet influences theproduction of volatile fatty acids which in turn modulate milkproduction, body condition, growth, etc. Reproduced from Moran, 2005.Tropical dairy farming: feeding management for small holder dairyfarmers in the humic tropics (Chapter 5), Landlinks Press, 312 pp.

FIG. 4 depicts the large change in microbial alpha-diversity as ananimal continues to be fed a finishing diet.

FIG. 5 depicts the importance of the speed of microbial successions intoa streamlined microbial ensemble related to animal efficiency.Furthermore, the large shift in the rumen microbiome on week 4 is mostsignificant when related to efficiency.

FIG. 6 depicts the average ruminal VFA concentrations of high-RFI andlow-RFI steers. More efficient animals tended to have a higherproduction of VFAs.

FIG. 7 depicts the theoretical concentrations of dissolved CO2 withrespect to rumen pH. Reproduced from Laporte-Uribe, 2016.

FIG. 8 depicts the theorized pH of the rumen based on both VFAconcentrations and dCO₂ concentrations (filled squares), and VFAconcentrations alone (open squares). Including dCO₂ better predicts pHthan VFAs alone. Reproduced from Laporte-Uribe, 2016.

FIG. 9 depicts the potential path ruminal CO2 may follow to impact thephysiology of a ruminant. Reproduced from Laporte-Uribe, 2016.

FIG. 10A and FIG. 10B depict the phylogenetic diversity of rumenbacterial communities across samples at week 1 and week 5 of the study(FIG. 10A) and across samples at week 7 and week 10 of the study (FIG.10B).

FIG. 11A and FIG. 11B depict the machine learning prediction accuracyutilized in the study. The residual feed intake (RFI) is predictive ofserum metabolic signature (FIG. 11A) and the RFI is predictive of rumenmicrobiome signature (FIG. 11B).

FIG. 12 depicts the mean predictive compounds between low-RFI andhigh-RFI. The greater the abundance of glucose-1-phosphate and/orglucose-6-phosphate is indicative of high-RFI.

FIG. 13 depicts the serum metabolic signatures correlated to week 10bacterial community composition.

FIG. 14A and FIG. 14B depict the week 10 rumen microbial communitycorrelations to serum metabolome. The mean serum pantothenate abundancediffers between low-RFI and high-RFI steers (FIG. 14A). The meanFlavobacteria abundance is associated with high pantothenate abundance(FIG. 14B).

FIG. 15 depicts spectral coclustering of composite average of OTU overtime, indicating major microbial successions throughout the study in therumen microbial community.

FIG. 16 depicts a principal coordinate analysis (PCoA) based onBray-Curtis distances among the ruminal bacterial communities throughoutthe study.

FIG. 17 depicts the relative abundance of three bacterial orders drivingthe shift in bacterial community composition throughout the 10-weektrial. Shaded regions indicate SEM. These three order are important toRFI. A major microbial succession occurs in week 4.

FIG. 18A and FIG. 18B depicts the microbial differentiation over pHbetween animals on week 5 following the large microbial succession inthe rumen on week 4. Rumen alpha-diversity correlated with pH (PearsonR=0.44; P=0.0051) (FIG. 18A). Rumen pH is predictive of bacterialcommunity structure at week 5 (R²=0.48; P=0.04) (FIG. 18B). PC=PrincipleComponent.

FIG. 19 depicts the plot of the residual feed intake (RFI) of the 50steers.

FIG. 20 depicts a principal coordinate analysis (PCoA) of all samplesthroughout the study. Each dot represents a rumen sample's bacterialcommunity, and the distance between dots represents the differencebetween those communities. The shading of each dot represents the timein weeks when the sample was taken.

FIG. 21 depicts a Kullback-Leibler (K-L) divergence. The K-L divergenceis a method for determining the commotional distance between two sets(i.e., difference between two sample's microbial communities). In FIG.21 reveals the comparison as to how much the microbial community changesweek to week between high RFI animals and low RFI animals. The figureindicates that in week 4 the amount of change in the microbialcommunities is important to the RFI.

FIG. 22 depicts the AUC for the residual feed intake. Random Forestsmachine learning was used to predict the RFI on any given week given themicrobial community composition. The microbial community is mostpredictive of RFI at week four.

FIG. 23 depicts the abundance of three taxonomic orders by weekseparated by RFI, which indicates the taxa that are having a directcorrelation with RFI and at what time. The figure shows that week fouris important for Aeromonadales and Pasteurellales populations inrelation to RFI.

FIG. 24 depicts a flow chart for the synthesis of Vitamin B orpantothenate, and the intermediate products produced to arrive atAcyl-CoA species. Reproduced from Basu and Blair. 2012. NatureProtocols. 7:1-11.

FIG. 25 depicts the evaluation of the microbes of the present disclosureand their ability to modulate weight in the animal, particularly theability to increase the weight of the animal.

FIG. 26 depicts the evaluation of the microbes of the present disclosureand their ability to modulate pH in the rumen, particularly the abilityto increase the pH in the rumen while the animal is being fed a highgrain diet.

FIG. 27 depicts the evaluation of the microbes of the present disclosureand their ability to decrease CO2 in the rumen.

FIG. 28 depicts the evaluation of the microbes of the present disclosureand their ability to modulate CO2 in the rumen, particularly the abilityto decrease CO2 in the rumen while the animal is being fed a high graindiet.

FIG. 29 depicts the MIC score distribution for common performanceparameters including average daily weight gain, weight gain, feedintake, and feed efficiency with six species of bacteria, in which manyof the species have been evaluated in 3^(rd) party studies. The lowerthe MIC score, the less likely the species/strains associated with thatMIC score are capable of positively modulating the aforementionedperformance parameters.

FIG. 30 depicts the KEGG carbon dioxide fixation pathways inprokaryotes.

FIG. 31 depicts the PATRIC carbon dioxide fixation pathways inprokaryotes.

FIG. 32 depicts the feed conversion ratio (FCR) for animals thatreceived microbes vs. animals that did not receive microbes.

DETAILED DESCRIPTION Definitions

While the following terms are believed to be well understood by one ofordinary skill in the art, the following definitions are set forth tofacilitate explanation of the presently disclosed subject matter.

The term “a” or “an” may refer to one or more of that entity, i.e. canrefer to plural referents. As such, the terms “a” or “an”, “one or more”and “at least one” are used interchangeably herein. In addition,reference to “an element” by the indefinite article “a” or “an” does notexclude the possibility that more than one of the elements is present,unless the context clearly requires that there is one and only one ofthe elements.

Reference throughout this specification to “one embodiment”, “anembodiment”, “one aspect”, or “an aspect” means that a particularfeature, structure or characteristic described in connection with theembodiment is included in at least one embodiment of the presentdisclosure. Thus, the appearances of the phrases “in one embodiment” or“in an embodiment” in various places throughout this specification arenot necessarily all referring to the same embodiment. Furthermore, theparticular features, structures, or characteristics can be combined inany suitable manner in one or more embodiments.

As used herein, in particular embodiments, the terms “about” or“approximately” when preceding a numerical value indicates the valueplus or minus a range of 10%.

As used herein the terms “microorganism” or “microbe” should be takenbroadly. These terms are used interchangeably and include, but are notlimited to, the two prokaryotic domains, Bacteria and Archaea,eukaryotic fungi and protozoa, as well as viruses. In some embodiments,the disclosure refers to the “microbes” of Table 1 and/or Table 2, orthe “microbes” incorporated by reference. This characterization canrefer to not only the predicted taxonomic microbial identifiers of thetable, but also the identified strains of the microbes listed in thetable.

The term “microbial community” means a group of microbes comprising twoor more species or strains. Unlike microbial ensemble, a microbialcommunity does not have to be carrying out a common function, or doesnot have to be participating in, or leading to, or correlating with, arecognizable parameter, such as a phenotypic trait of interest (e.g.increased feed efficiency in beef cattle).

As used herein, “isolate,” “isolated,” “isolated microbe,” and liketerms, are intended to mean that the one or more microorganisms has beenseparated from at least one of the materials with which it is associatedin a particular environment (for example soil, water, animal tissue).

Microbes of the present disclosure may include spores and/or vegetativecells. In some embodiments, microbes of the present disclosure includemicrobes in a viable but non-culturable (VBNC) state, or a quiescentstate. See Liao and Zhao (US Publication US2015267163A1). In someembodiments, microbes of the present disclosure include microbes in abiofilm. See Merritt et al. (U.S. Pat. No. 7,427,408).

Thus, an “isolated microbe” does not exist in its naturally occurringenvironment; rather, it is through the various techniques describedherein that the microbe has been removed from its natural setting andplaced into a non-naturally occurring state of existence. Thus, theisolated strain or isolated microbe may exist as, for example, abiologically pure culture, or as spores (or other forms of the strain)in association with an acceptable carrier.

As used herein, “spore” or “spores” refer to structures produced bybacteria and fungi that are adapted for survival and dispersal. Sporesare generally characterized as dormant structures; however, spores arecapable of differentiation through the process of germination.Germination is the differentiation of spores into vegetative cells thatare capable of metabolic activity, growth, and reproduction. Thegermination of a single spore results in a single fungal or bacterialvegetative cell. Fungal spores are units of asexual reproduction, and insome cases are necessary structures in fungal life cycles. Bacterialspores are structures for surviving conditions that may ordinarily benonconductive to the survival or growth of vegetative cells.

As used herein, “microbial composition” refers to a compositioncomprising one or more microbes of the present disclosure, wherein amicrobial composition, in some embodiments, is administered to animalsof the present disclosure.

As used herein, “carrier”, “acceptable carrier”, or “pharmaceuticalcarrier” refers to a diluent, adjuvant, excipient, or vehicle with whichthe compound is administered. Such carriers can be sterile liquids, suchas water and oils, including those of petroleum, animal, vegetable, orsynthetic origin; such as peanut oil, soybean oil, mineral oil, sesameoil, and the like. Water or aqueous solution saline solutions andaqueous dextrose and glycerol solutions are preferably employed ascarriers, in some embodiments as injectable solutions. In someembodiments, gelling agents are employed as carriers. Alternatively, thecarrier can be a solid dosage form carrier, including but not limited toone or more of a binder (for compressed pills), a glidant, anencapsulating agent, a flavorant, and a colorant. The choice of carriercan be selected with regard to the intended route of administration andstandard pharmaceutical practice. See Hardee and Baggo (1998.Development and Formulation of Veterinary Dosage Forms. 2^(nd) Ed. CRCPress. 504 pg.); E. W. Martin (1970. Remington's PharmaceuticalSciences. 17^(th) Ed. Mack Pub. Co.); and Blaser et al. (US PublicationUS20110280840A1).

In some aspects, carriers may be granular in structure, such as sand orsand particles. In further aspects, the carriers may be dry, as opposedto a moist or wet carrier. In some aspects, carriers can be nutritivesubstances and/or prebiotic substances selected fromfructo-oligosaccharides, inulins, isomalto-oligosaccharides, lactitol,lactosucruse, lactulose, pyrodextrines, soy oligosaccharides,transgalacto-oligosaccharides, xylo-oligosaccharides, trace minerals,and vitamins. In some aspects, carriers can be in solid or liquid form.In some aspects, carriers can be zeolites, calcium carbonate, magnesiumcarbonate, silicon dioxide, ground corn, trehalose, chitosan, shellac,albumin, starch, skim-milk powder, sweet-whey powder, maltodextrin,lactose, and inulin. In some aspects, a carrier is water orphysiological saline.

The term “bioensemble,” “microbial ensemble,” or “synthetic ensemble”refers to a composition comprising one or more active microbesidentified by methods, systems, and/or apparatuses of the presentdisclosure and that do not naturally exist in a naturally occurringenvironment and/or at ratios or amounts that do not exist in nature. Abioensemble is a subset of a microbial community of individual microbialspecies, or strains of a species, which can be described as carrying outa common function, or can be described as participating in, or leadingto, or correlating with, a recognizable parameter, such as a phenotypictrait of interest (e.g. increased feed efficiency in feedlot cattle).The bioensemble may comprise two or more species, or strains of aspecies, of microbes. In some instances, the microbes coexist within thecommunity symbiotically.

In certain aspects of the disclosure, the isolated microbes exist asisolated and biologically pure cultures. It will be appreciated by oneof skill in the art, that an isolated and biologically pure culture of aparticular microbe, denotes that said culture is substantially free(within scientific reason) of other living organisms and contains onlythe individual microbe in question. The culture can contain varyingconcentrations of said microbe. The present disclosure notes thatisolated and biologically pure microbes often “necessarily differ fromless pure or impure materials.” See, e.g. In re Bergstrom, 427 F.2d1394, (CCPA 1970)(discussing purified prostaglandins), see also, In reBergy, 596 F.2d 952 (CCPA 1979)(discussing purified microbes), see also,Parke-Davis & Co. v. H. K. Mulford & Co., 189 F. 95 (S.D.N.Y. 1911)(Learned Hand discussing purified adrenaline), aff'd in part, rev′d inpart, 196 F. 496 (2d Cir. 1912), each of which are incorporated hereinby reference. Furthermore, in some aspects, the disclosure provides forcertain quantitative measures of the concentration, or puritylimitations, that must be found within an isolated and biologically puremicrobial culture. The presence of these purity values, in certainembodiments, is a further attribute that distinguishes the presentlydisclosed microbes from those microbes existing in a natural state. See,e.g., Merck & Co. v. Olin Mathieson Chemical Corp., 253 F.2d 156 (4thCir. 1958) (discussing purity limitations for vitamin B12 produced bymicrobes), incorporated herein by reference.

As used herein, “individual isolates” should be taken to mean acomposition, or culture, comprising a predominance of a single genera,species, or strain, of microorganism, following separation from one ormore other microorganisms. The phrase should not be taken to indicatethe extent to which the microorganism has been isolated or purified.However, “individual isolates” can comprise substantially only onegenus, species, or strain, of microorganism.

As used herein, “microbiome” refers to the collection of microorganismsthat inhabit the digestive tract or gastrointestinal tract of an animal(including the rumen if said animal is a ruminant) and themicroorganism's physical environment (i.e. the microbiome has a bioticand physical component). The microbiome is fluid and may be modulated bynumerous naturally occurring and artificial conditions (e.g., change indiet, disease, antimicrobial agents, influx of additionalmicroorganisms, etc.). The modulation of the microbiome of a rumen thatcan be achieved via administration of the compositions of thedisclosure, can take the form of: (a) increasing or decreasing aparticular Family, Genus, Species, or functional grouping of microbe(i.e. alteration of the biotic component of the rumen microbiome) and/or(b) increasing or decreasing volatile fatty acids in the rumen,increasing or decreasing rumen pH, increasing or decreasing any otherphysical parameter important for rumen health (i.e. alteration of theabiotic component of the rumen microbiome).

As used herein, “probiotic” refers to a substantially pure microbe(i.e., a single isolate) or a mixture of desired microbes, and may alsoinclude any additional components that can be administered to beefcattle for restoring microbiota. Probiotics or microbial inoculantcompositions of the disclosure may be administered with an agent toallow the microbes to survive the environment of the gastrointestinaltract, i.e., to resist low pH and to grow in the gastrointestinalenvironment. In some embodiments, the present compositions (e.g.,microbial compositions) are probiotics in some aspects.

As used herein, “prebiotic” refers to an agent that increases the numberand/or activity of one or more desired microbes. Non-limiting examplesof prebiotics that may be useful in the methods of the presentdisclosure include fructooligosaccharides (e.g., oligofructose, inulin,inulin-type fructans), galactooligosaccharides, amino acids, alcohols,and mixtures thereof. See Ramirez-Farias et al. (2008. Br. J. Nutr.4:1-10) and Pool-Zobel and Sauer (2007. J. Nutr. 137:2580-2584 andsupplemental).

The term “growth medium” as used herein, is any medium which is suitableto support growth of a microbe. By way of example, the media may benatural or artificial including gastrin supplemental agar, LB media,blood serum, and tissue culture gels. It should be appreciated that themedia may be used alone or in combination with one or more other media.It may also be used with or without the addition of exogenous nutrients.

The term “relative abundance” as used herein, is the number orpercentage of a microbe present in the gastrointestinal tract or otherorgan system, relative to the number or percentage of total microbespresent in said tract or organ system. The relative abundance may alsobe determined for particular types of microbes such as bacteria, fungi,viruses, and/or protozoa, relative to the total number or percentage ofbacteria, fungi, viruses, and/or protozoa present. In one embodiment,relative abundance is determined by PCR. In another embodiment, relativeabundance is determined by colony forming unit assays (cfu) or plaqueforming unit assays (pfu) performed on samples from the gastrointestinaltract or other organ system of interest.

The medium may be amended or enriched with additional compounds orcomponents, for example, a component which may assist in the interactionand/or selection of specific groups of microorganisms. For example,antibiotics (such as penicillin) or sterilants (for example, quaternaryammonium salts and oxidizing agents) could be present and/or thephysical conditions (such as salinity, nutrients (for example organicand inorganic minerals (such as phosphorus, nitrogenous salts, ammonia,potassium and micronutrients such as cobalt and magnesium), pH, and/ortemperature), methionine, prebiotics, ionophores, and beta glucans couldbe amended.

As used herein, the term “ruminant” includes mammals that are capable ofacquiring nutrients from plant-based food by fermenting it in aspecialized stomach (rumen) prior to digestion, principally throughmicrobial actions. Ruminants included cattle, goats, sheep, giraffes,yaks, deer, antelope, and others.

As used herein, the term “bovid” includes any member of family Bovidae,which include hoofed mammals such as antelope, sheep, goats, and cattle,among others.

As used herein, the term “steer” includes any member, species, variant,or hybrid of Bos indicus, Bos taurus indicus, or Bos taurus taurus. Theterm “steer” further includes reference to cow (mature female), steer(castrated male), heifer (immature female not having born offspring),bull (mature uncastrated male), and calve (immature males or females).

As used herein, the terms “beef cattle” and “feedlot cattle” are usedsynonymously to refer to cattle that are grown and utilized for theproduction of beef. Said cattle of the present disclosure includevarieties such as the following: Africander, Angus, Aubrac, Barzona,Bazadaise, Beef Shorthorn, Beefalo, Beefmaster, Belgian Blue, BelmontRed, Belted Galloway, Black Angus, Blonde d'Aquitaine, Bonsmara, Boran,Bradford, Brahman, Brahmousin, Brangus, British White, Buelingo,Canchim, Caracu, Charolais, Chianina, Composite, Corriente, Devon,Dexter, Drakensberger, Droughtmaster, English Longhorn, Galloway,Gelbvieh, Gloucester, Hays Converter, Hereford, Highland, Holstein,Hybridmaster, Limousin, Lincoln Red, Lowline, Luing, Maine-Anjou, Rougedes Pres, Marchigiana, Miniature Hereford, Mirandesa, Mongolian, MurrayGrey, Nelore, Nguni, Parthenais, Piemontese, Pinzgauer, Red Angus, RedPoll, Retinta, Romagnola, Salers, Sanganer, Santa Cruz, Santa Gertrudis,Senepol, Shetland, Simbrah, Simmental, South Devon, Speckle Park, SquareMeaters, Sussex, Tarentaise, Texas Longhorn, Tuli, Wagyu, Watusi, WelshBlack, Whitebred Shorthorn, and Zebu; or hybrids and/or crosses thereof.

As used herein, “dairy cattle” or “dairy cows” are used synonymously torefer to cows that are grown and utilized for the production of milk.

As used herein, “performance” should be taken to be increased weightgain, improved feed efficiency, improved residual feed intake, improvedfeed intake.

As used herein, “improved” should be taken broadly to encompassimprovement of a characteristic of interest, as compared to a controlgroup, or as compared to a known average quantity associated with thecharacteristic in question. For example, “improved” feed efficiencyassociated with application of a beneficial microbe, or microbialensemble, of the disclosure can be demonstrated by comparing the feedefficiency of beef cattle treated by the microbes taught herein to thefeed efficiency of beef cattle not treated. In the present disclosure,“improved” does not necessarily demand that the data be statisticallysignificant (i.e. p<0.05); rather, any quantifiable differencedemonstrating that one value (e.g. the average treatment value) isdifferent from another (e.g. the average control value) can rise to thelevel of “improved.”

As used herein, “inhibiting and suppressing” and like terms should notbe construed to require complete inhibition or suppression, althoughthis may be desired in some embodiments.

The term “marker” or “unique marker” as used herein is an indicator ofunique microorganism type, microorganism strain or activity of amicroorganism strain. A marker can be measured in biological samples andincludes without limitation, a nucleic acid-based marker such as aribosomal RNA gene, a peptide- or protein-based marker, and/or ametabolite or other small molecule marker.

The term “metabolite” as used herein is an intermediate or product ofmetabolism. A metabolite in one embodiment is a small molecule.Metabolites have various functions, including in fuel, structural,signaling, stimulatory and inhibitory effects on enzymes, as a cofactorto an enzyme, in defense, and in interactions with other organisms (suchas pigments, odorants and pheromones). A primary metabolite is directlyinvolved in normal growth, development and reproduction. A secondarymetabolite is not directly involved in these processes but usually hasan important ecological function. Examples of metabolites include butare not limited to antibiotics and pigments such as resins and terpenes,etc. Some antibiotics use primary metabolites as precursors, such asactinomycin which is created from the primary metabolite, tryptophan.Metabolites, as used herein, include small, hydrophilic carbohydrates;large, hydrophobic lipids and complex natural compounds.

As used herein, the term “genotype” refers to the genetic makeup of anindividual cell, cell culture, tissue, organism, or group of organisms.

As used herein, the term “allele(s)” means any of one or morealternative forms of a gene, all of which alleles relate to at least onetrait or characteristic. In a diploid cell, the two alleles of a givengene occupy corresponding loci on a pair of homologous chromosomes.Since the present disclosure, in embodiments, relates to QTLs, i.e.genomic regions that may comprise one or more genes or regulatorysequences, it is in some instances more accurate to refer to “haplotype”(i.e. an allele of a chromosomal segment) instead of “allele”, however,in those instances, the term “allele” should be understood to comprisethe term “haplotype”. Alleles are considered identical when they expressa similar phenotype. Differences in sequence are possible but notimportant as long as they do not influence phenotype.

As used herein, the term “locus” (loci plural) means a specific place orplaces or a site on a chromosome where for example a gene or geneticmarker is found.

As used herein, the term “genetically linked” refers to two or moretraits that are co-inherited at a high rate during breeding such thatthey are difficult to separate through crossing.

A “recombination” or “recombination event” as used herein refers to achromosomal crossing over or independent assortment. The term“recombinant” refers to an organism having a new genetic makeup arisingas a result of a recombination event.

As used herein, the term “molecular marker” or “genetic marker” refersto an indicator that is used in methods for visualizing differences incharacteristics of nucleic acid sequences. Examples of such indicatorsare restriction fragment length polymorphism (RFLP) markers, amplifiedfragment length polymorphism (AFLP) markers, single nucleotidepolymorphisms (SNPs), insertion mutations, microsatellite markers(SSRs), sequence-characterized amplified regions (SCARs), cleavedamplified polymorphic sequence (CAPS) markers or isozyme markers orcombinations of the markers described herein which defines a specificgenetic and chromosomal location. Markers further include polynucleotidesequences encoding 16S or 18S rRNA, and internal transcribed spacer(ITS) sequences, which are sequences found between small-subunit andlarge-subunit rRNA genes that have proven to be especially useful inelucidating relationships or distinctions among when compared againstone another. Mapping of molecular markers in the vicinity of an alleleis a procedure which can be performed by the average person skilled inmolecular-biological techniques.

The primary structure of major rRNA subunit 16S comprise a particularcombination of conserved, variable, and hypervariable regions thatevolve at different rates and enable the resolution of both very ancientlineages such as domains, and more modern lineages such as genera. Thesecondary structure of the 16S subunit include approximately 50 heliceswhich result in base pairing of about 67% of the residues. These highlyconserved secondary structural features are of great functionalimportance and can be used to ensure positional homology in multiplesequence alignments and phylogenetic analysis. Over the previous fewdecades, the 16S rRNA gene has become the most sequenced taxonomicmarker and is the cornerstone for the current systematic classificationof bacteria and archaea (Yarza et al. 2014. Nature Rev. Micro.12:635-45).

A sequence identity of 94.5% or lower for two 16S rRNA genes is strongevidence for distinct genera, 86.5% or lower is strong evidence fordistinct families, 82% or lower is strong evidence for distinct orders,78.5% is strong evidence for distinct classes, and 75% or lower isstrong evidence for distinct phyla. The comparative analysis of 16S rRNAgene sequences enables the establishment of taxonomic thresholds thatare useful not only for the classification of cultured microorganismsbut also for the classification of the many environmental sequences.Yarza et al. 2014. Nature Rev. Micro. 12:635-45).

As used herein, the term “trait” refers to a characteristic orphenotype. For example, in the context of some embodiments of thepresent disclosure; efficiency of feed utilization, particularly withcorn-intensive diets; amount of feces produced; susceptibility to gutpathogens; and a decrease in mortality rates; among others. Desirabletraits may also include other characteristics, including but not limitedto: an increase in weight; an increase in average daily weight gain; anincrease of musculature; an increase of fatty acid concentration in thegastrointestinal tract; an improved efficiency in feed utilization anddigestibility; an increase in polysaccharide and lignin degradation; anincrease in fat, starch, and/or protein digestion; an increase in fattyacid concentration in the rumen; pH balance in the rumen, an increase invitamin availability; an increase in mineral availability; an increasein amino acid availability; a reduction in methane and/or nitrous oxideemissions; a reduction in manure production; an improved dry matterintake; an improved efficiency of nitrogen utilization; an improvedefficiency of phosphorous utilization; an increased resistance tocolonization of pathogenic microbes that colonize cattle; reducedmortality; increased production of antimicrobials; increased clearanceof pathogenic microbes; increased resistance to colonization ofpathogenic microbes that colonize cattle; increased resistance tocolonization of pathogenic microbes that infect humans; reducedincidence of acidosis or bloat; increased meat marbling, increased ordecreased red coloring of meat, increased or decreasedtexture/coarseness of meat; increased amount of USDA Prime, USDA Choice,and USDA Select quality meat per animal, increased in the number ofanimals producing USDA Prime, USDA Choice, and USDA Select quality meat;increase or reduced concentration or presence of volatile compounds inthe meat; reduced prevalence of acidosis or bloat; reduced bodytemperature; and any combination thereof; wherein said increase orreduction is determined by comparing against an animal not having beenadministered said composition.

A trait may be inherited in a dominant or recessive manner, or in apartial or incomplete-dominant manner. A trait may be monogenic (i.e.determined by a single locus) or polygenic (i.e. determined by more thanone locus) or may also result from the interaction of one or more geneswith the environment.

In the context of this disclosure, traits may also result from theinteraction of one or more beef cattle genes and one or moremicroorganism genes.

As used herein, the term “homozygous” means a genetic condition existingwhen two identical alleles reside at a specific locus, but arepositioned individually on corresponding pairs of homologous chromosomesin the cell of a diploid organism. Conversely, as used herein, the term“heterozygous” means a genetic condition existing when two differentalleles reside at a specific locus, but are positioned individually oncorresponding pairs of homologous chromosomes in the cell of a diploidorganism.

As used herein, the term “phenotype” refers to the observablecharacteristics of an individual cell, cell culture, organism (e.g.,cattle), or group of organisms which results from the interactionbetween that individual's genetic makeup (i.e., genotype) and theenvironment.

As used herein, the term “chimeric” or “recombinant” when describing anucleic acid sequence or a protein sequence refers to a nucleic acid, ora protein sequence, that links at least two heterologouspolynucleotides, or two heterologous polypeptides, into a singlemacromolecule, or that re-arranges one or more elements of at least onenatural nucleic acid or protein sequence. For example, the term“recombinant” can refer to an artificial combination of two otherwiseseparated segments of sequence, e.g., by chemical synthesis or by themanipulation of isolated segments of nucleic acids by geneticengineering techniques.

As used herein, a “synthetic nucleotide sequence” or “syntheticpolynucleotide sequence” is a nucleotide sequence that is not known tooccur in nature or that is not naturally occurring. Generally, such asynthetic nucleotide sequence will comprise at least one nucleotidedifference when compared to any other naturally occurring nucleotidesequence.

As used herein, the term “nucleic acid” refers to a polymeric form ofnucleotides of any length, either ribonucleotides ordeoxyribonucleotides, or analogs thereof. This term refers to theprimary structure of the molecule, and thus includes double- andsingle-stranded DNA, as well as double- and single-stranded RNA. It alsoincludes modified nucleic acids such as methylated and/or capped nucleicacids, nucleic acids containing modified bases, backbone modifications,and the like. The terms “nucleic acid” and “nucleotide sequence” areused interchangeably.

As used herein, the term “gene” refers to any segment of DNA associatedwith a biological function. Thus, genes include, but are not limited to,coding sequences and/or the regulatory sequences required for theirexpression. Genes can also include non-expressed DNA segments that, forexample, form recognition sequences for other proteins. Genes can beobtained from a variety of sources, including cloning from a source ofinterest or synthesizing from known or predicted sequence information,and may include sequences designed to have desired parameters.

As used herein, the term “homologous” or “homologue” or “ortholog” isknown in the art and refers to related sequences that share a commonancestor or family member and are determined based on the degree ofsequence identity. The terms “homology,” “homologous,” “substantiallysimilar” and “corresponding substantially” are used interchangeablyherein. They refer to nucleic acid fragments wherein changes in one ormore nucleotide bases do not affect the ability of the nucleic acidfragment to mediate gene expression or produce a certain phenotype.These terms also refer to modifications of the nucleic acid fragments ofthe instant disclosure such as deletion or insertion of one or morenucleotides that do not substantially alter the functional properties ofthe resulting nucleic acid fragment relative to the initial, unmodifiedfragment. It is therefore understood, as those skilled in the art willappreciate, that the disclosure encompasses more than the specificexemplary sequences. These terms describe the relationship between agene found in one species, subspecies, variety, cultivar or strain andthe corresponding or equivalent gene in another species, subspecies,variety, cultivar or strain. For purposes of this disclosure homologoussequences are compared. “Homologous sequences” or “homologues” or“orthologs” are thought, believed, or known to be functionally related.A functional relationship may be indicated in any one of a number ofways, including, but not limited to: (a) degree of sequence identityand/or (b) the same or similar biological function. Preferably, both (a)and (b) are indicated. Homology can be determined using softwareprograms readily available in the art, such as those discussed inCurrent Protocols in Molecular Biology (F. M. Ausubel et al., eds.,1987) Supplement 30, section 7.718, Table 7.71. Some alignment programsare MacVector (Oxford Molecular Ltd, Oxford, U.K.), ALIGN Plus(Scientific and Educational Software, Pennsylvania) and AlignX (VectorNTI, Invitrogen, Carlsbad, Calif.). Another alignment program isSequencher (Gene Codes, Ann Arbor, Mich.), using default parameters.

As used herein, the term “nucleotide change” refers to, e.g., nucleotidesubstitution, deletion, and/or insertion, as is well understood in theart. For example, mutations contain alterations that produce silentsubstitutions, additions, or deletions, but do not alter the propertiesor activities of the encoded protein or how the proteins are made.

As used herein, the term “protein modification” refers to, e.g., aminoacid substitution, amino acid modification, deletion, and/or insertion,as is well understood in the art.

As used herein, the term “at least a portion” or “fragment” of a nucleicacid or polypeptide means a portion having the minimal sizecharacteristics of such sequences, or any larger fragment of the fulllength molecule, up to and including the full length molecule. Afragment of a polynucleotide of the disclosure may encode a biologicallyactive portion of a genetic regulatory element. A biologically activeportion of a genetic regulatory element can be prepared by isolating aportion of one of the polynucleotides of the disclosure that comprisesthe genetic regulatory element and assessing activity as describedherein. Similarly, a portion of a polypeptide may be 4 amino acids, 5amino acids, 6 amino acids, 7 amino acids, and so on, going up to thefull length polypeptide. The length of the portion to be used willdepend on the particular application. A portion of a nucleic acid usefulas a hybridization probe may be as short as 12 nucleotides; in someembodiments, it is 20 nucleotides. A portion of a polypeptide useful asan epitope may be as short as 4 amino acids. A portion of a polypeptidethat performs the function of the full-length polypeptide wouldgenerally be longer than 4 amino acids.

Variant polynucleotides also encompass sequences derived from amutagenic and recombinogenic procedure such as DNA shuffling. Strategiesfor such DNA shuffling are known in the art. See, for example, Stemmer(1994) PNAS 91:10747-10751; Stemmer (1994) Nature 370:389-391; Crameriet al. (1997) Nature Biotech. 15:436-438; Moore et al. (1997) J. Mol.Biol. 272:336-347; Zhang et al. (1997) PNAS 94:4504-4509; Crameri et al.(1998) Nature 391:288-291; and U.S. Pat. Nos. 5,605,793 and 5,837,458.For PCR amplifications of the polynucleotides disclosed herein,oligonucleotide primers can be designed for use in PCR reactions toamplify corresponding DNA sequences from cDNA or genomic DNA extractedfrom any organism of interest. Methods for designing PCR primers and PCRcloning are generally known in the art and are disclosed in Sambrook etal. (1989) Molecular Cloning: A Laboratory Manual (2nd ed., Cold SpringHarbor Laboratory Press, Plainview, N.Y.). See also Innis et al., eds.(1990) PCR Protocols: A Guide to Methods and Applications (AcademicPress, New York); Innis and Gelfand, eds. (1995) PCR Strategies(Academic Press, New York); and Innis and Gelfand, eds. (1999) PCRMethods Manual (Academic Press, New York). Known methods of PCR include,but are not limited to, methods using paired primers, nested primers,single specific primers, degenerate primers, gene-specific primers,vector-specific primers, partially-mismatched primers, and the like.

The term “primer” as used herein refers to an oligonucleotide which iscapable of annealing to the amplification target allowing a DNApolymerase to attach, thereby serving as a point of initiation of DNAsynthesis when placed under conditions in which synthesis of primerextension product is induced, i.e., in the presence of nucleotides andan agent for polymerization such as DNA polymerase and at a suitabletemperature and pH. The (amplification) primer is preferably singlestranded for maximum efficiency in amplification. Preferably, the primeris an oligodeoxyribonucleotide. The primer must be sufficiently long toprime the synthesis of extension products in the presence of the agentfor polymerization. The exact lengths of the primers will depend on manyfactors, including temperature and composition (A/T vs. G/C content) ofprimer. A pair of bi-directional primers consists of one forward and onereverse primer as commonly used in the art of DNA amplification such asin PCR amplification.

The terms “stringency” or “stringent hybridization conditions” refer tohybridization conditions that affect the stability of hybrids, e.g.,temperature, salt concentration, pH, formamide concentration and thelike. These conditions are empirically optimized to maximize specificbinding and minimize non-specific binding of primer or probe to itstarget nucleic acid sequence. The terms as used include reference toconditions under which a probe or primer will hybridize to its targetsequence, to a detectably greater degree than other sequences (e.g. atleast 2-fold over background). Stringent conditions are sequencedependent and will be different in different circumstances. Longersequences hybridize specifically at higher temperatures. Generally,stringent conditions are selected to be about 5° C. lower than thethermal melting point (Tm) for the specific sequence at a defined ionicstrength and pH. The Tm is the temperature (under defined ionic strengthand pH) at which 50% of a complementary target sequence hybridizes to aperfectly matched probe or primer. Typically, stringent conditions willbe those in which the salt concentration is less than about 1.0 MNa+ion, typically about 0.01 to 1.0 M Na+ion concentration (or othersalts) at pH 7.0 to 8.3 and the temperature is at least about 30° C. forshort probes or primers (e.g. 10 to 50 nucleotides) and at least about60° C. for long probes or primers (e.g. greater than 50 nucleotides).Stringent conditions may also be achieved with the addition ofdestabilizing agents such as formamide. Exemplary low stringentconditions or “conditions of reduced stringency” include hybridizationwith a buffer solution of 30% formamide, 1 M NaCl, 1% SDS at 37° C. anda wash in 2×SSC at 40° C. Exemplary high stringency conditions includehybridization in 50% formamide, 1M NaCl, 1% SDS at 37° C., and a wash in0.1×SSC at 60° C. Hybridization procedures are well known in the art andare described by e.g. Ausubel et al., 1998 and Sambrook et al., 2001. Insome embodiments, stringent conditions are hybridization in 0.25 MNa2HPO4 buffer (pH 7.2) containing 1 mM Na2EDTA, 0.5-20% sodium dodecylsulfate at 45° C., such as 0.5%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%,10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19% or 20%, followed by awash in 5×SSC, containing 0.1% (w/v) sodium dodecyl sulfate, at 55° C.to 65° C.

As used herein, “promoter” refers to a DNA sequence capable ofcontrolling the expression of a coding sequence or functional RNA. Thepromoter sequence consists of proximal and more distal upstreamelements, the latter elements often referred to as enhancers.Accordingly, an “enhancer” is a DNA sequence that can stimulate promoteractivity, and may be an innate element of the promoter or a heterologouselement inserted to enhance the level or tissue specificity of apromoter. Promoters may be derived in their entirety from a native gene,or be composed of different elements derived from different promotersfound in nature, or even comprise synthetic DNA segments. It isunderstood by those skilled in the art that different promoters maydirect the expression of a gene in different tissues or cell types, orat different stages of development, or in response to differentenvironmental conditions. It is further recognized that since in mostcases the exact boundaries of regulatory sequences have not beencompletely defined, DNA fragments of some variation may have identicalpromoter activity.

As used herein, a “constitutive promoter” is a promoter which is activeunder most conditions and/or during most development stages. There areseveral advantages to using constitutive promoters in expression vectorsused in biotechnology, such as: high level of production of proteinsused to select transgenic cells or organisms; high level of expressionof reporter proteins or scorable markers, allowing easy detection andquantification; high level of production of a transcription factor thatis part of a regulatory transcription system; production of compoundsthat requires ubiquitous activity in the organism; and production ofcompounds that are required during all stages of development.Non-limiting exemplary constitutive promoters include, CaMV 35Spromoter, opine promoters, ubiquitin promoter, alcohol dehydrogenasepromoter, etc.

As used herein, a “non-constitutive promoter” is a promoter which isactive under certain conditions, in certain types of cells, and/orduring certain development stages. For example, tissue specific, tissuepreferred, cell type specific, cell type preferred, inducible promoters,and promoters under development control are non-constitutive promoters.Examples of promoters under developmental control include promoters thatpreferentially initiate transcription in certain tissues.

As used herein, “inducible” or “repressible” promoter is a promoterwhich is under chemical or environmental factors control. Examples ofenvironmental conditions that may affect transcription by induciblepromoters include anaerobic conditions, certain chemicals, the presenceof light, acidic or basic conditions, etc.

As used herein, a “tissue specific” promoter is a promoter thatinitiates transcription only in certain tissues. Unlike constitutiveexpression of genes, tissue-specific expression is the result of severalinteracting levels of gene regulation. As such, in the art sometimes itis preferable to use promoters from homologous or closely relatedspecies to achieve efficient and reliable expression of transgenes inparticular tissues. This is one of the main reasons for the large amountof tissue-specific promoters isolated from particular tissues found inboth scientific and patent literature.

As used herein, the term “operably linked” refers to the association ofnucleic acid sequences on a single nucleic acid fragment so that thefunction of one is regulated by the other. For example, a promoter isoperably linked with a coding sequence when it is capable of regulatingthe expression of that coding sequence (i.e., that the coding sequenceis under the transcriptional control of the promoter). Coding sequencescan be operably linked to regulatory sequences in a sense or antisenseorientation. In another example, the complementary RNA regions of thedisclosure can be operably linked, either directly or indirectly, 5′ tothe target mRNA, or 3′ to the target mRNA, or within the target mRNA, ora first complementary region is 5′ and its complement is 3′ to thetarget mRNA.

As used herein, the phrases “recombinant construct”, “expressionconstruct”, “chimeric construct”, “construct”, and “recombinant DNAconstruct” are used interchangeably herein. A recombinant constructcomprises an artificial combination of nucleic acid fragments, e.g.,regulatory and coding sequences that are not found together in nature.For example, a chimeric construct may comprise regulatory sequences andcoding sequences that are derived from different sources, or regulatorysequences and coding sequences derived from the same source, butarranged in a manner different than that found in nature. Such constructmay be used by itself or may be used in conjunction with a vector. If avector is used then the choice of vector is dependent upon the methodthat will be used to transform host cells as is well known to thoseskilled in the art. For example, a plasmid vector can be used. Theskilled artisan is well aware of the genetic elements that must bepresent on the vector in order to successfully transform, select andpropagate host cells comprising any of the isolated nucleic acidfragments of the disclosure. The skilled artisan will also recognizethat different independent transformation events will result indifferent levels and patterns of expression (Jones et al., (1985) EMBOJ. 4:2411-2418; De Almeida et al., (1989) Mol. Gen. Genetics 218:78-86),and thus that multiple events must be screened in order to obtain linesdisplaying the desired expression level and pattern. Such screening maybe accomplished by Southern analysis of DNA, Northern analysis of mRNAexpression, immunoblotting analysis of protein expression, or phenotypicanalysis, among others. Vectors can be plasmids, viruses,bacteriophages, pro-viruses, phagemids, transposons, artificialchromosomes, and the like, that replicate autonomously or can integrateinto a chromosome of a host cell. A vector can also be a naked RNApolynucleotide, a naked DNA polynucleotide, a polynucleotide composed ofboth DNA and RNA within the same strand, a poly-lysine-conjugated DNA orRNA, a peptide-conjugated DNA or RNA, a liposome-conjugated DNA, or thelike, that is not autonomously replicating. As used herein, the term“expression” refers to the production of a functional end-product e.g.,an mRNA or a protein (precursor or mature).

In some embodiments, the cell or organism has at least one heterologoustrait. As used herein, the term “heterologous trait” refers to aphenotype imparted to a transformed host cell or transgenic organism byan exogenous DNA segment, heterologous polynucleotide or heterologousnucleic acid. Various changes in phenotype are of interest to thepresent disclosure, including but not limited to increasing yield of aneconomically important trait (e.g., weight, etc.) and the like. Theseresults can be achieved by providing expression of heterologous productsor increased expression of endogenous products in organisms using themethods and compositions of the present disclosure.

As used herein, the term “MIC” means maximal information coefficient.MIC is a type of nonparamentric network analysis that identifies a score(MIC score) between active microbial strains of the present disclosureand at least one measured metadata (e.g., milk fat). Further, U.S.application Ser. No. 15/217,575, filed on Jul. 22, 2016 (issued as U.S.Pat. No. 9,540,676 on Jan. 10, 2017) is hereby incorporated by referencein its entirety.

The maximal information coefficient (MIC) is then calculated betweenstrains and metadata 3021a, and between strains 3021b; as seen in FIG. 2. Results are pooled to create a list of all relationships and theircorresponding MIC scores 3022. If the relationship scores below a giventhreshold 3023, the relationship is deemed/identified as irrelevant3023b. If the relationship is above a given threshold 3023, therelationship deemed/identified as relevant 2023a, and is further subjectto network analysis 3024. The following code fragment shows an exemplarymethodology for such analysis, according to one embodiment:

Read total list of relationships file as links threshold = 0.8 for i inrange(len(links)):  if links >= threshold   multiplier[i] = 1  else  multiplier[i] = 0 end if links_temp = multiplier*links final_links =links_temp[links_temp != 0] savetxt(output_file,final_links)output_file.close( )

In some embodiments, the compositions of the present disclosure compriseone or more bacteria and/or one or more fungi that have a MIC score ofat least about 0.1, at least about 0.15, at least about 0.2, at leastabout 0.25, at least about 0.3, at least about 0.35, at least about 0.4,at least about 0.45, at least about 0.5, at least about 0.55, at leastabout 0.6, at least about 0.65, at least about 0.7, at least about 0.75,at least about 0.80, at least about 0.85, at least about 0.9, or atleast about 0.95.

In some embodiments, the compositions of the present disclosure compriseone or more bacteria and/or one or more fungi that have a MIC score ofat least 0.1, at least 0.15, at least 0.2, at least 0.25, at least 0.3,at least 0.35, at least 0.4, at least 0.45, at least 0.5, at least 0.55,at least 0.6, at least 0.65, at least 0.7, at least 0.75, at least 0.80,at least 0.85, at least 0.9, or at least 0.95.

Based on the output of the network analysis, active strains are selected3025 for preparing products (e.g., ensembles, aggregates, and/or othersynthetic groupings) containing the selected strains. The output of thenetwork analysis can also be used to inform the selection of strains forfurther product composition testing.

The use of thresholds is discussed above for analyses anddeterminations. Thresholds can be, depending on the implementation andapplication: (1) empirically determined (e.g., based on distributionlevels, setting a cutoff at a number that removes a specified orsignificant portion of low level reads); (2) any non-zero value; (3)percentage/percentile based; (4) only strains whose normalized secondmarker (i.e., activity) reads is greater than normalized first marker(cell count) reads; (5) log 2 fold change between activity and quantityor cell count; (6) normalized second marker (activity) reads is greaterthan mean second marker (activity) reads for entire sample (and/orsample set); and/or any magnitude threshold described above in additionto a statistical threshold (i.e., significance testing). The followingexample provides thresholding detail for distributions of RNA-basedsecond marker measurements with respect to DNA-based first markermeasurements, according to one embodiment.

As used herein “shelf-stable” refers to a functional attribute and newutility acquired by the microbes formulated according to the disclosure,which enable said microbes to exist in a useful/active state outside oftheir natural environment in the rumen (i.e. a markedly differentcharacteristic). Thus, shelf-stable is a functional attribute created bythe formulations/compositions of the disclosure and denoting that themicrobe formulated into a shelf-stable composition can exist outside therumen and under ambient conditions for a period of time that can bedetermined depending upon the particular formulation utilized, but ingeneral means that the microbes can be formulated to exist in acomposition that is stable under ambient conditions for at least a fewdays and generally at least one week. Accordingly, a “shelf-stableruminant supplement” is a composition comprising one or more microbes ofthe disclosure, said microbes formulated in a composition, such that thecomposition is stable under ambient conditions for at least one week,meaning that the microbes comprised in the composition (e.g. whole cell,spore, or lysed cell) are able to impart one or more beneficialphenotypic properties to a ruminant when administered (e.g. increasedmilk yield, improved milk compositional characteristics, improved rumenhealth, and/or modulation of the rumen microbiome).

Feedlot Cattle vs. Dairy Cows

The instant subject matter is distinct over the subject matter of priorAscus Biosciences, Inc. applications. The 16S sequences of the microbesof the instant disclosure are believed to be distinct over those of anyprior Ascus Biosciences, Inc. applications. One of ordinary skill in theart would be aware that the diet of a dairy cow would be distinct fromthat of a steer on a beef feedlot. The steer on the beef feedlot wouldbe fed a high-energy high-grain diet in order to quickly increase therate of weight gain and to increase the maximum weight prior torendering. The cow on the dairy farm would be fed a different diet thatis optimized for the production of milk with little consideration forrapid weight gain or highest maximum weight. The two diets would resultin the rumen of the animals in the two environments to yield drasticallydifferent microbiota. Thus, the microorganisms in the rumen of the dairycow and that of the feedlot steer are expected to be different from oneanother.

Isolated Microbes

In some aspects, the present disclosure provides isolated microbes,including novel strains of microbes, presented in Table 1 and Table 2.

In other aspects, the present disclosure provides isolated wholemicrobial cultures of the microbes identified in Table 1 and Table 2.These cultures may comprise microbes at various concentrations.

In some aspects, the disclosure provides for utilizing one or moremicrobes selected from Table 1 and Table 2 to increase a phenotypictrait of interest in beef cattle.

In some embodiments, the disclosure provides isolated microbial speciesbelonging to taxonomic families of Prevotellaceae, Veillonellaceae,Ruminococcaceae, Fibrobacteraceae, Bacillaceae 1, Spirochaetaceae,Bacteroidaceae, Lachnospiraceae, Porphyromonadaceae, Coriobacteriaceae,Acidaminococcaceae, Clostridiaceae 1, Rhodobacteraceae, Cryomorphaceae,Erysipelotrichaceae, Promicromonosporaceae, Burkholderiaceae,Succinivibrionaceae, Pseudomonadaceae, Corynebacteriaceae,Planococcaceae, Streptomycetaceae, Synergistaceae, Nocardiopsaceae,Flavobacteriaceae, Propionibacteriaceae, Staphylococcaceae,Clostridiales incertae sedis XIII, Anaeroplasmataceae, Pasteurellaceae,Caulobacteraceae, and Sphingomonadaceae.

In further embodiments, isolated microbial species may be selected fromgenera of Fibrobacter, Saccharofermentans, Bacillus, Spirochaeta,Bacteroides, Lachnospiracea incertae sedis, Clostridium XLVa,Ruminococcus, Butyricimonas, Olsenella, Acidaminococcus,Parabacteroides, Clostridum sensu stricto, Oribacterium,Pseudoflavonifractor, Treponema, Rhodobacter, Fluviicola,Succiniclasticum, Solobacterium, Veillonella, Cellulosimicrobium,Cupriavidus, Megasphaera, Succinivibrio, Oscillibacter, Pseudomonas,Corynebacterium, Adlercreutzia, Dorea, Roseburia, Anaerovibrio,Sporosarcina, Streptomyces, Syntrophococcus, Butyrivibrio,Lachnobacterium, Pyramidobacter, Coprococcus, Ruminobacter,Thermobifidia, Papillibacter, Aquimarina, Propioniciclava,Staphylococcus, Mogibacterium, Pseudobutyrivibrio, Asteroleplasma,Turicibacter, Aggregatibacter, Brevundimonas, Phascolarctobacterium, andSphingobium.

Furthermore, the disclosure relates to microbes having characteristicssubstantially similar to that of a microbe identified in Table 1 and/orTable 2.

The isolated microbial species, and novel strains of said species,identified in the present disclosure, are able to impart beneficialproperties or traits to beef cattle production.

For instance, the isolated microbes described in Table 1 and Table 2, ormicrobial ensemble of said microbes, are able to increase feedefficiency. The increase can be quantitatively measured, for example, bymeasuring the effect that said microbial application has upon themodulation of feed efficiency.

In some embodiments, the isolated microbial strains are microbes of thepresent disclosure that have been genetically modified. In someembodiments, the genetically modified or recombinant microbes comprisepolynucleotide sequences which do not naturally occur in said microbes.In some embodiments, the microbes may comprise heterologouspolynucleotides. In further embodiments, the heterologouspolynucleotides may be operably linked to one or more polynucleotidesnative to the microbes.

In some embodiments, the heterologous polynucleotides may be reportergenes or selectable markers. In some embodiments, reporter genes may beselected from any of the family of fluorescence proteins (e.g., GFP,RFP, YFP, and the like), β-galactosidase, luciferase. In someembodiments, selectable markers may be selected from neomycinphosphotransferase, hygromycin phosphotransferase, aminoglycosideadenyltransferase, dihydrofolate reductase, acetolactase synthase,bromoxynil nitrilase, β-glucuronidase, dihydrogolate reductase, andchloramphenicol acetyltransferase. In some embodiments, the heterologouspolynucleotide may be operably linked to one or more promoter.

In some embodiments the isolated microbial strains express transgenic ornative polypeptides selected from cellulases (endocellulases,exocellulases, glucosidases), pectinases, amylases, amylopectinases,ligninases, and phytases.

The isolated microbes of Table 2 represent variants of the microbesrecited in Table 1. The microbes of Table 2, comprise the referencestrains, and the microbes of Table 1 comprise the variants thereof.Generally, the variants of Table 2 comprise 16S rRNA sequences thatshare at least about 97% sequence identity with the 16S rRNA of thecorresponding reference strain in Table B. For example, strainsAsbusbbf_873A to Asbusbbf_873G (Table 2 correspond to variants of thereference strain Ascusbbf_873 (Table 1), wherein the 16S rRNA of thevariants share at least about 97% sequence identity with that of thereference strain.

Microbial Compositions

In some aspects, the disclosure provides microbial compositionscomprising a combination of at least any two microbes selected fromamongst the microbes identified in Table 1 and Table 2.

In certain embodiments, the compositions of the present disclosurecomprise two microbes, or three microbes, or four microbes, or fivemicrobes, or six microbes, or seven microbes, or eight microbes, or ninemicrobes, or ten or more microbes. Said microbes of the compositions aredifferent microbial species, or different strains of a microbialspecies.

In some embodiments, the disclosure provides microbial compositions,comprising: at least one or at least two isolated microbial speciesbelonging to genera of: Fibrobacter, Saccharofermentans, Bacillus,Spirochaeta, Bacteroides, Lachnospiracea incertae sedis, ClostridiumXLVa, Ruminococcus, Butyricimonas, Olsenella, Acidaminococcus,Parabacteroides, Clostridum sensu stricto, Oribacterium,Pseudoflavonifractor, Treponema, Rhodobacter, Fluviicola,Succiniclasticum, Solobacterium, Veillonella, Cellulosimicrobium,Cupriavidus, Megasphaera, Succinivibrio, Oscillibacter, Pseudomonas,Corynebacterium, Adlercreutzia, Dorea, Roseburia, Anaerovibrio,Sporosarcina, Streptomyces, Syntrophococcus, Butyrivibrio,Lachnobacterium, Pyramidobacter, Coprococcus, Ruminobacter,Thermobifidia, Papillibacter, Aquimarina, Propioniciclava,Staphylococcus, Mogibacterium, Pseudobutyrivibrio, Asteroleplasma,Turicibacter, Aggregatibacter, Brevundimonas, Phascolarctobacterium, andSphingobium. Particular novel strains of species of these aforementionedgenera can be found in Table 1 and Table 2.

In some embodiments, the disclosure provides microbial compositions,comprising: at least one or at least two isolated microbial speciesbelonging to the family of: Prevotellaceae, Veillonellaceae,Ruminococcaceae, Fibrobacteraceae, Bacillaceae 1, Spirochaetaceae,Bacteroidaceae, Lachnospiraceae, Porphyromonadaceae, Coriobacteriaceae,Acidaminococcaceae, Clostridiaceae 1, Rhodobacteraceae, Cryomorphaceae,Erysipelotrichaceae, Promicromonosporaceae, Burkholderiaceae,Succinivibrionaceae, Pseudomonadaceae, Corynebacteriaceae,Planococcaceae, Streptomycetaceae, Synergistaceae, Nocardiopsaceae,Flavobacteriaceae, Propionibacteriaceae, Staphylococcaceae,Clostridiales incertae sedis XIII, Anaeroplasmataceae, Pasteurellaceae,Caulobacteraceae, and Sphingomonadaceae.

Particular novel strains of species of these aforementioned genera canbe found in Table 1 and Table 2.

In particular aspects, the disclosure provides microbial compositions,comprising species as grouped in Tables 3-9. With respect to Tables 3-9,the letters A through I represent a non-limiting selection of microbesof the present disclosure, defined as:

A=Strain designation Ascusbbf_154 identified in Table 1;

B=Strain designation Ascusbbf_4 identified in Table 1;

C=Strain designation Ascusbbf_14146 identified in Table 1;

D=Strain designation Ascusbbf_876 identified in Table 1;

E=Strain designation Ascusbbf_24302 identified in Table 1;

F=Strain designation Ascusbbf_1085 identified in Table 1;

G=Strain designation Ascusbbf_1 identified in Table 1;

H=Strain designation Ascusbbf_6176 identified in Table 1; and

I=Strain designation Ascusbbf_3427 identified in Table 1.

TABLE 3 Eight and Nine Strain Compositions A, B, C, D, E, F, G, H A, B,C, D, E, F, G, I A, B, C, D, E, F, H, I A, B, C, D, E, G, H, I A, B, C,D, F, G, H, I A, B, C, E, F, G, H, I A, B, D, E, F, G, H, I A, C, D, E,F, G, H, I B, C, D, E, F, G, H, I A, B, C, D, E, F, G, H, I

TABLE 4 Seven Strain Compositions A, B, C, D, E, F, G A, B, C, D, E, F,H A, B, C, D, E, F, I A, B, C, D, E, G, H A, B, C, D, E, G, I A, B, C,D, E, H, I A, B, C, D, F, G, H A, B, C, D, F, G, I A, B, C, D, F, H, IA, B, C, D, G, H, I A, B, C, E, F, G, H A, B, C, E, F, G, I A, B, C, E,F, H, I A, B, C, E, G, H, I A, B, C, F, G, H, I A, B, D, E, F, G, H A,B, D, E, F, G, I A, B, D, E, F, H, I A, B, D, E, G, H, I A, B, D, F, G,H, I A, B, E, F, G, H, I A, C, D, E, F, G, H A, C, D, E, F, G, I A, C,D, E, F, H, I A, C, D, E, G, H, I A, C, D, F, G, H, I A, C, E, F, G, H,I A, D, E, F, G, H, I B, C, D, E, F, G, H B, C, D, E, F, G, I B, C, D,E, F, H, I B, C, D, E, G, H, I B, C, D, F, G, H, I B, C, E, F, G, H, IB, D, E, F, G, H, I C, D, E, F, G, H, I

TABLE 5 Six Strain Compositions A, B, C, D, E, F A, B, C, D, E, G A, B,C, D, E, H A, B, C, D, E, I A, B, C, D, F, G A, B, C, D, F, H A, B, C,D, F, I A, B, C, D, G, H A, B, C, D, G, I A, B, C, D, H, I A, B, C, E,F, G A, B, C, E, F, H A, B, C, E, F, I A, B, C, E, G, H A, B, C, E, G, IA, B, C, E, H, I A, B, C, F, G, H A, B, C, F, G, I A, B, C, F, H, I A,B, C, G, H, I A, B, D, E, F, G A, B, D, E, F, H A, B, D, E, F, I A, B,D, E, G, H A, B, D, E, G, I A, B, D, E, H, I A, B, D, F, G, H A, B, D,F, G, I D, E, F, G, H, I C, E, F, G, H, I A, B, D, F, H, I A, B, D, G,H, I A, B, E, F, G, H A, B, E, F, G, I A, B, E, F, H, I A, B, E, G, H, IA, B, F, G, H, I A, C, D, E, F, G A, C, D, E, F, H A, C, D, E, F, I A,C, D, E, G, H A, C, D, E, G, I A, C, D, E, H, I A, C, D, F, G, H A, C,D, F, G, I A, C, D, F, H, I A, C, D, G, H, I A, C, E, F, G, H A, C, E,F, G, I A, C, E, F, H, I A, C, E, G, H, I A, C, F, G, H, I A, D, E, F,G, H A, D, E, F, G, I A, D, E, F, H, I A, D, E, G, H, I A, D, F, G, H, IA, E, F, G, H, I B, C, D, E, F, G B, C, D, E, F, H B, C, D, E, F, I B,C, D, E, G, H B, C, D, E, G, I B, C, D, E, H, I B, C, D, F, G, H B, C,D, F, G, I B, C, D, F, H, I B, C, D, G, H, I B, C, E, F, G, H B, C, E,F, G, I B, C, E, F, H, I B, C, E, G, H, I B, C, F, G, H, I B, D, E, F,G, H B, D, E, F, G, I B, D, E, F, H, I B, D, E, G, H, I B, D, F, G, H, IB, E, F, G, H, I C, D, E, F, G, H C, D, E, F, G, I C, D, E, F, H, I C,D, E, G, H, I C, D, F, G, H, I

TABLE 6 Five Strain Compositions A, B, C, D, E A, B, C, D, F A, B, C, D,G A, B, C, D, H A, B, C, D, I A, B, C, E, F A, B, C, E, G A, B, C, E, HA, B, C, F, H A, B, C, F, G A, B, C, F, I A, B, C, G, H A, B, C, G, I A,B, C, H, I A, B, D, E, F A, B, D, E, G A, B, D, E, I A, B, D, F, G A, B,D, F, H A, B, D, F, I A, B, D, G, H A, B, D, G, I A, B, D, H, I A, B, E,F, G A, B, E, F, I A, B, E, G, H A, B, E, G, I A, B, E, H, I A, B, F, G,H A, B, F, G, I A, B, F, H, I A, B, G, H, I A, C, D, E, G A, C, D, E, HA, C, D, E, I A, C, D, F, G A, C, D, F, H A, C, D, F, I A, C, D, G, H A,C, D, G, I A, C, E, F, G A, C, E, F, H A, C, E, F, I A, C, E, G, H A, C,E, G, I A, C, E, H, I A, C, F, G, H A, C, F, G, I A, C, G, H, I A, D, E,F, G A, D, E, F, H A, D, E, F, I A, D, E, G, H A, D, E, G, I A, D, E, H,I A, D, F, G, H A, D, F, H, I A, D, G, H, I A, E, F, G, H A, E, F, G, IA, E, F, H, I A, E, G, H, I A, F, G, H, I B, C, D, E, F B, C, D, E, H B,C, D, E, I B, C, D, F, G B, C, D, F, H B, C, D, F, I B, C, D, G, H B, C,D, G, I B, C, D, H, I B, C, E, F, H B, C, E, F, I B, C, E, G, H B, C, E,G, I B, C, E, H, I B, C, F, G, H B, C, F, G, I B, C, F, H, I B, D, E, F,G B, D, E, F, H B, D, E, F, I B, D, E, G, H B, D, E, G, I B, D, E, H, IB, D, F, G, H B, D, F, G, I B, D, G, H, I B, E, F, G, H B, E, F, G, I B,E, F, H, I B, E, G, H, I B, F, G, H, I C, D, E, F, G C, D, E, F, H C, D,E, G, H C, D, E, G, I C, D, E, H, I C, D, F, G, H C, D, F, G, I C, D, F,H, I C, D, G, H, I C, E, F, G, H C, E, F, H, I C, E, G, H, I C, F, G, H,I D, E, F, G, H D, E, F, G, I D, E, F, H, I D, E, G, H, I D, F, G, H, IA, B, C, E, I A, B, D, E, H A, B, E, F, H A, C, D, E, F A, C, D, H, I A,C, F, H, I A, D, F, G, I B, C, D, E, G B, C, E, F, G B, C, G, H, I B, D,F, H, I C, D, E, F, I C, E, F, G, I E, F, G, H, I

TABLE 7 Four Strain Compositions A, B, C, D A, B, C, E A, B, C, F A, B,C, G A, B, C, H A, B, C, I A, B, D, E A, B, D, F D, G, H, I A, B, D, GA, B, D, H A, B, D, I A, B, E, F A, B, E, G A, B, E, H A, B, E, I A, B,F, G E, F, G, H A, B, F, H A, D, F, H A, D, F, I A, D, G, H A, D, G, IA, D, H, I A, E, F, G A, E, F, H E, F, G, I A, B, F, I A, B, G, H A, B,G, I A, B, H, I A, C, D, E A, C, D, F A, C, D, G A, C, D, H E, F, H, IA, C, D, I A, C, E, F A, C, E, G A, C, E, H A, C, E, I A, C, F, G A, C,F, H A, C, F, I E, G, H, I A, C, G, H A, C, G, I A, C, H, I A, D, E, FA, D, E, G A, D, E, H A, D, E, I A, D, F, G F, G, H, I A, E, F, I A, E,G, H A, E, G, I A, E, H, I A, F, G, H A, F, G, I A, F, H, I A, G, H, ID, E, F, H B, C, D, E B, C, D, F B, C, D, G B, C, D, H B, C, D, I B, C,E, F B, C, E, G B, C, E, H D, E, F, I B, C, E, I B, C, F, G B, C, F, HB, C, F, I B, C, G, H B, C, G, I B, C, H, I B, D, E, F D, E, G, H B, D,E, G B, D, E, H B, D, E, I B, D, F, G B, D, F, H B, D, F, I B, D, G, HB, D, G, I D, E, G, I B, D, H, I B, E, F, G B, E, F, H B, E, F, I B, E,G, H B, E, G, I B, E, H, I B, F, G, H D, E, H, I B, F, G, I B, F, H, IB, G, H, I C, D, E, F C, D, E, G C, D, E, H C, D, E, I C, D, F, G D, F,G, H C, D, F, H C, D, F, I C, D, G, H C, D, G, I C, D, H, I C, E, F, GC, E, F, H C, E, F, I D, F, G, I C, E, G, H C, E, G, I C, E, H, I C, F,G, H C, F, G, I C, F, H, I C, G, H, I D, E, F, G D, F, H, I

TABLE 8 Three Strain Compositions A, B, C A, B, D A, B, E A, B, F A, B,G A, B, H A, B, I A, C, D A, C, E G, H, I E, F, H A, C, F A, C, G A, C,H A, C, I A, D, E A, D, F A, D, G A, D, H A, D, I F, H, I E, F, G A, E,F A, E, G A, E, H A, E, I A, F, G A, F, H A, F, I A, G, H A, G, I F, G,I D, H, I A, H, I B, C, D B, C, E B, C, F B, C, G B, C, H B, C, I B, D,E B, D, F F, G, H D, G, I B, D, G B, D, H B, D, I B, E, F B, E, G B, E,H B, E, I B, F, G B, F, H E, H, I E, F, I B, F, I B, G, H B, G, I B, H,I C, D, E C, D, F C, D, G C, D, H C, D, I E, G, I D, G, H C, E, F C, E,G C, E, H C, E, I C, F, G C, F, H C, F, I C, G, H C, G, I E, G, H D, F,I C, H, I D, E, F D, E, G D, E, H D, E, I D, F, G D, F, H

TABLE 9 Two Strain Compositions A, B A, C A, D A, E A, F A, G A, H A, IB, C B, D B, E B, F B, G B, H B, I C, D C, E C, F C, G C, H C, I D, E D,F D, G D, H D, I E, F E, G E, H E, I F, G F, H F, I G, H G, I H, I

In some embodiments, the microbial compositions may be selected from anymember group from Tables 3-9.

Isolated Microbes—Source Material

In particular embodiments, the microbes of the present compositions arenot naturally found in association with the same animal. In someaspects, the microbial species forming the microbial community are allfound in association with animals from the same geographic location. Inother aspects, each microbial species forming the composition is from adifferent geographic location. A geographic location can be definedbased upon the predominant soil type in a region, the predominantclimate in a region, the predominant plant community present in aregion, the predominant plant community present in a region, thedistance between regions, the average rainfall in a region, amongothers.

In some embodiments, the microbes of the present compositions are notnaturally found in association with the same species of animal. In someembodiments, the microbes of the present compositions are found in thesame species of animal, but separated by geographic region.

In a particular embodiment, at least one microbial species that is amember of the microbial community derived by the disclosed method isnative to, or was acquired from, a geographic region at least about 1 m,10 m, 100 m, 1 km, 10 km, 100 km, 1,000 km, 10,000 km, 20,000 km, 30,000km, or 40,000 km from the location of the plant upon which a phenotypictrait is to be increased based upon the taught methods.

The microbes of the present disclosure were obtained, among otherplaces, at various locales in the United States from thegastrointestinal tract of beef cattle.

Isolated Microbes—Microbial Culture Techniques

The microbes of Table 1 and Table 2 were matched to their nearesttaxonomic groups by utilizing classification tools of the RibosomalDatabase Project (RDP) for 16s rRNA sequences and the User-friendlyNordic ITS Ectomycorrhiza (UNITE) database for ITS rRNA sequences.Examples of matching microbes to their nearest taxa may be found in Lanet al. (2012. PLOS one. 7(3):e32491), Schloss and Westcott (2011. Appl.Environ. Microbiol. 77(10):3219-3226), and Koljalg et al. (2005. NewPhytologist. 166(3):1063-1068).

The isolation, identification, and culturing of the microbes of thepresent disclosure can be effected using standard microbiologicaltechniques. Examples of such techniques may be found in Gerhardt, P.(ed.) Methods for General and Molecular Microbiology. American Societyfor Microbiology, Washington, D.C. (1994) and Lennette, E. H. (ed.)Manual of Clinical Microbiology, Third Edition. American Society forMicrobiology, Washington, D.C. (1980), each of which is incorporated byreference.

Isolation can be effected by streaking the specimen on a solid medium(e.g., nutrient agar plates) to obtain a single colony, which ischaracterized by the phenotypic traits described hereinabove (e.g., Grampositive/negative, capable of forming spores aerobically/anaerobically,cellular morphology, carbon source metabolism, acid/base production,enzyme secretion, metabolic secretions, etc.) and to reduce thelikelihood of working with a culture which has become contaminated.

For example, for microbes of the disclosure, biologically pure isolatescan be obtained through repeated subculture of biological samples, eachsubculture followed by streaking onto solid media to obtain individualcolonies or colony forming units. Methods of preparing, thawing, andgrowing lyophilized bacteria are commonly known, for example, Gherna, R.L. and C. A. Reddy. 2007. Culture Preservation, p 1019-1033. In C. A.Reddy, T. J. Beveridge, J. A. Breznak, G. A. Marzluf, T. M. Schmidt, andL. R. Snyder, eds. American Society for Microbiology, Washington, D.C.,1033 pages; herein incorporated by reference. Thus freeze dried liquidformulations and cultures stored long term at −70° C. in solutionscontaining glycerol are contemplated for use in providing formulationsof the present disclosure.

The microbes of the disclosure can be propagated in a liquid mediumunder aerobic conditions, or alternatively anaerobic conditions. Mediumfor growing the bacterial strains of the present disclosure includes acarbon source, a nitrogen source, and inorganic salts, as well asspecially required substances such as vitamins, amino acids, nucleicacids and the like. Examples of suitable carbon sources which can beused for growing the microbes include, but are not limited to, starch,peptone, yeast extract, amino acids, sugars such as glucose, arabinose,mannose, glucosamine, maltose, and the like; salts of organic acids suchas acetic acid, fumaric acid, adipic acid, propionic acid, citric acid,gluconic acid, malic acid, pyruvic acid, malonic acid and the like;alcohols such as ethanol and glycerol and the like; oil or fat such assoybean oil, rice bran oil, olive oil, corn oil, sesame oil. The amountof the carbon source added varies according to the kind of carbon sourceand is typically between 1 to 100 gram(s) per liter of medium.Preferably, glucose, starch, and/or peptone is contained in the mediumas a major carbon source, at a concentration of 0.1-5% (W/V). Examplesof suitable nitrogen sources which can be used for growing the bacterialstrains of the present disclosure include, but are not limited to, aminoacids, yeast extract, tryptone, beef extract, peptone, potassiumnitrate, ammonium nitrate, ammonium chloride, ammonium sulfate, ammoniumphosphate, ammonia or combinations thereof. The amount of nitrogensource varies according to the type of nitrogen source, typicallybetween 0.1 to 30 gram per liter of medium. The inorganic salts,potassium dihydrogen phosphate, dipotassium hydrogen phosphate, disodiumhydrogen phosphate, magnesium sulfate, magnesium chloride, ferricsulfate, ferrous sulfate, ferric chloride, ferrous chloride, manganoussulfate, manganous chloride, zinc sulfate, zinc chloride, cupricsulfate, calcium chloride, sodium chloride, calcium carbonate, sodiumcarbonate can be used alone or in combination. The amount of inorganicacid varies according to the kind of the inorganic salt, typicallybetween 0.001 to 10 gram per liter of medium. Examples of speciallyrequired substances include, but are not limited to, vitamins, nucleicacids, yeast extract, peptone, meat extract, malt extract, dried yeastand combinations thereof. Cultivation can be effected at a temperature,which allows the growth of the microbial strains, essentially, between20° C. and 46° C. In some aspects, a temperature range is 30° C.-39° C.For optimal growth, in some embodiments, the medium can be adjusted topH 6.0-7.4. It will be appreciated that commercially available media mayalso be used to culture the microbial strains, such as Nutrient Broth orNutrient Agar available from Difco, Detroit, Mich. It will beappreciated that cultivation time may differ depending on the type ofculture medium used and the concentration of sugar as a major carbonsource.

In some aspects, cultivation lasts between 24-96 hours. Microbial cellsthus obtained are isolated using methods, which are well known in theart. Examples include, but are not limited to, membrane filtration andcentrifugal separation. The pH may be adjusted using sodium hydroxideand the like and the culture may be dried using a freeze dryer, untilthe water content becomes equal to 4% or less. Microbial co-cultures maybe obtained by propagating each strain as described hereinabove. In someaspects, microbial multi-strain cultures may be obtained by propagatingtwo or more of the strains described hereinabove. It will be appreciatedthat the microbial strains may be cultured together when compatibleculture conditions can be employed.

Isolated Microbes—Microbial Strains

Microbes can be distinguished into a genus based on polyphasic taxonomy,which incorporates all available phenotypic and genotypic data into aconsensus classification (Vandamme et al. 1996. Polyphasic taxonomy, aconsensus approach to bacterial systematics. Microbiol Rev 1996,60:407-438). One accepted genotypic method for defining species is basedon overall genomic relatedness, such that strains which shareapproximately 70% or more relatedness using DNA-DNA hybridization, with5° C. or less ΔT_(m) (the difference in the melting temperature betweenhomologous and heterologous hybrids), under standard conditions, areconsidered to be members of the same species. Thus, populations thatshare greater than the aforementioned 70% threshold can be considered tobe variants of the same species. Another accepted genotypic method fordefining species is to isolate marker genes of the present disclosure,sequence these genes, and align these sequenced genes from multipleisolates or variants. The microbes are interpreted as belonging to thesame species if one or more of the sequenced genes share at least 97%sequence identity.

The 16S or 18S rRNA sequences or ITS sequences are often used for makingdistinctions between species and strains, in that if one of theaforementioned sequences shares less than a specified % sequenceidentity from a reference sequence, then the two organisms from whichthe sequences were obtained are said to be of different species orstrains.

Thus, one could consider microbes to be of the same species, if theyshare at least 80%, 85%, 90%, 95%, 97%, 98%, or 99% sequence identityacross the 16S or 18S rRNA sequence, or the ITS1 or ITS2 sequence.

Further, one could define microbial strains of a species, as those thatshare at least 80%, 85%, 90%, 95%, 97%, 98%, or 99% sequence identityacross the 16S or 18S rRNA sequence, or the ITS1 or ITS2 sequence.

In one embodiment, microbial strains of the present disclosure includethose that comprise polynucleotide sequences that share at least 70%,75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%,93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity with any oneof SEQ ID NOs:1-5,993. In a further embodiment, microbial strains of thepresent disclosure include those that comprise polynucleotide sequencesthat share at least 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%,88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%sequence identity with any one of SEQ ID NOs:1-5,993.

Comparisons may also be made with 23S rRNA sequences against referencesequences.

Unculturable microbes often cannot be assigned to a definite species inthe absence of a phenotype determination, the microbes can be given acandidatus designation within a genus provided their 16S or 18S rRNAsequences or ITS sequences subscribes to the principles of identity withknown species.

One approach is to observe the distribution of a large number of strainsof closely related species in sequence space and to identify clusters ofstrains that are well resolved from other clusters. This approach hasbeen developed by using the concatenated sequences of multiple core(house-keeping) genes to assess clustering patterns, and has been calledmultilocus sequence analysis (MLSA) or multilocus sequence phylogeneticanalysis. MLSA has been used successfully to explore clustering patternsamong large numbers of strains assigned to very closely related speciesby current taxonomic methods, to look at the relationships between smallnumbers of strains within a genus, or within a broader taxonomicgrouping, and to address specific taxonomic questions. More generally,the method can be used to ask whether bacterial species exist—that is,to observe whether large populations of similar strains invariably fallinto well-resolved clusters, or whether in some cases there is a geneticcontinuum in which clear separation into clusters is not observed.

In order to more accurately make a determination of genera, adetermination of phenotypic traits, such as morphological, biochemical,and physiological characteristics are made for comparison with areference genus archetype. The colony morphology can include color,shape, pigmentation, production of slime, etc. Features of the cell aredescribed as to shape, size, Gram reaction, extracellular material,presence of endospores, flagella presence and location, motility, andinclusion bodies. Biochemical and physiological features describe growthof the organism at different ranges of temperature, pH, salinity andatmospheric conditions, growth in presence of different sole carbon andnitrogen sources. One of ordinary skill in the art would be reasonablyapprised as to the phenotypic traits that define the genera of thepresent disclosure.

In one embodiment, the microbes taught herein were identified utilizing16S rRNA gene sequences and ITS sequences. It is known in the art that16S rRNA contains hypervariable regions that can providespecies/strain-specific signature sequences useful for bacterialidentification, and that ITS sequences can also providespecies/strain-specific signature sequences useful for fungalidentification.

Phylogenetic analysis using the rRNA genes and/or ITS sequences are usedto define “substantially similar” species belonging to common genera andalso to define “substantially similar” strains of a given taxonomicspecies. Furthermore, physiological and/or biochemical properties of theisolates can be utilized to highlight both minor and significantdifferences between strains that could lead to advantageous behavior inbeef cattle.

Compositions of the present disclosure may include combinations offungal spores and bacterial spores, fungal spores and bacterialvegetative cells, fungal vegetative cells and bacterial spores, fungalvegetative cells and bacterial vegetative cells. In some embodiments,compositions of the present disclosure comprise bacteria only in theform of spores. In some embodiments, compositions of the presentdisclosure comprise bacteria only in the form of vegetative cells. Insome embodiments, compositions of the present disclosure comprisebacteria in the absence of fungi. In some embodiments, compositions ofthe present disclosure comprise fungi in the absence of bacteria. Insome embodiments, compositions of the present disclosure comprise VBNCbacteria and/or fungi. In some embodiments, compositions of the presentdisclosure comprise bacteria and/or fungi in a quiescent state. In someembodiments, compositions of the present disclosure include dormantbacteria and/or fungi.

Bacterial spores may include endospores and akinetes. Fungal spores mayinclude statismospores, ballistospores, autospores, aplanospores,zoospores, mitospores, megaspores, microspores, meiospores,chlamydospores, urediniospores, teliospores, oospores, carpospores,tetraspores, sporangiospores, zygospores, ascospores, basidiospores,ascospores, and asciospores.

In some embodiments, spores of the composition germinate uponadministration to animals of the present disclosure. In someembodiments, spores of the composition germinate only uponadministration to animals of the present disclosure.

Microbial Compositions

In some embodiments, the microbes of the disclosure are combined intomicrobial compositions.

In some embodiments, the microbial compositions include cattle feed,such as grain and grain byproducts (barley, maize, oats, sorghum, wheat,distillers grains, sweet bran, and the like); roughage (alfalfa, silage,fescue, clover, ryegrass, and the like); starches (tapioca and thelike); protein (oilseed cakes, vegetable wastes, corn by-products, wheatby-products, and the like); liquid feeds (condensed corn distillerssolubles, molasses, tallow, yellow grease, corn oil, and the like); ornon-nitrogen protein. In some embodiments, the microbial compositionsinclude vitamins and/or metabolites thereof, minerals, urea, traceelements, emulsifiers, aromatizing products, binders, colorants,odorants, thickening agents, antibiotics, and the like. In someembodiments, the microbial compositions include one or more of anionophore; vaccine, antibiotic; antihelmintic; virucide; nematicide;amino acids such as methionine, glutamine, valine, glycine, cysteine,homocysteine, aspartic acid, and arginine; fish oil; oregano; carnitine,pantoate, pantothenate, aspartate, and biologically active moleculessuch as enzymes.

In some embodiments, the vitamins include vitamin B5, B1, B2, B3, B6,B9, B12, H, C, A, D, E, or K; and combinations thereof. In someembodiments, the microbial compositions include microbes that synthesizevitamin B5, B1, B2, B3, B6, B9, B12, H, C, A, D, E, and/or K. In someembodiments, the microbial compositions include microbes that synthesizevitamin B5. In some embodiments, the metabolites of vitamin B5, B1, B2,B3, B6, B9, B12, H, C, A, D, E, or K are contemplated as one or morecomponents of a microbial composition of the present disclosure. In oneembodiment, pantothenate is a component of a microbial composition ofthe present disclosure. In one embodiment, a component of a microbialcomposition of the present disclosure includes one or more precursorsutilized by mammalian or microbial biosynthesis of vitamins.

In some embodiments, the microbial compositions of the presentdisclosure are solid. Where solid compositions are used, it may bedesired to include one or more carrier materials including, but notlimited to: mineral earths such as silicas, talc, kaolin, limestone,chalk, clay, dolomite, diatomaceous earth; calcium sulfate; magnesiumsulfate; magnesium oxide; zeolites, calcium carbonate; magnesiumcarbonate; trehalose; chitosan; shellac; albumins; starch; skim milkpowder; sweet whey powder; maltodextrin; lactose; inulin; dextrose; andproducts of vegetable origin such as cereal meals, tree bark meal, woodmeal, and nutshell meal.

In some embodiments, the microbial compositions of the presentdisclosure are liquid. In further embodiments, the liquid comprises asolvent that may include water or an alcohol or a saline or carbohydratesolution, and other animal-safe solvents. In some embodiments, themicrobial compositions of the present disclosure include binders such asanimal-safe polymers, carboxymethylcellulose, starch, polyvinyl alcohol,and the like.

In some embodiments, the microbial compositions of the presentdisclosure comprise thickening agents such as silica, clay, naturalextracts of seeds or seaweed, synthetic derivatives of cellulose, guargum, locust bean gum, alginates, and methylcelluloses. In someembodiments, the microbial compositions comprise anti-settling agentssuch as modified starches, polyvinyl alcohol, xanthan gum, and the like.

In some embodiments, the microbial compositions of the presentdisclosure comprise colorants including organic chromophores classifiedas nitroso; nitro; azo, including monoazo, bisazo and polyazo; acridine,anthraquinone, azine, diphenylmethane, indamine, indophenol, methine,oxazine, phthalocyanine, thiazine, thiazole, triarylmethane, xanthene.In some embodiments, the microbial compositions of the presentdisclosure comprise trace nutrients such as salts of iron, manganese,boron, copper, cobalt, molybdenum and zinc. In some embodiments, themicrobial compositions comprise dyes, both natural and artificial. Insome embodiments, the dye is green in color.

In some embodiments, the microbial compositions of the presentdisclosure comprise an animal-safe virucide, parasiticide, bacteriocide,fungicide, or nematicide.

In some embodiments, microbial compositions of the present disclosurecomprise saccharides (e.g., monosaccharides, disaccharides,trisaccharides, polysaccharides, oligosaccharides, and the like),polymeric saccharides, lipids, polymeric lipids, lipopolysaccharides,proteins, polymeric proteins, lipoproteins, nucleic acids, nucleic acidpolymers, silica, inorganic salts and combinations thereof. In a furtherembodiment, microbial compositions comprise polymers of agar, agarose,gelrite, gellan gum, and the like. In some embodiments, microbialcompositions comprise plastic capsules, emulsions (e.g., water and oil),membranes, and artificial membranes. In some embodiments, emulsions orlinked polymer solutions may comprise microbial compositions of thepresent disclosure. See Harel and Bennett (U.S. Pat. No. 8,460,726 B2).

In some embodiments, microbial compositions of the present disclosurecomprise one or more exygen scavengers, denitrifies, nitrifiers, heavymetal chelators, and/or dechlorinators; and combinations thereof. In oneembodiment, the one or more exygen scavengers, denitrifiers, nitrifiers,heavy metal chelators, and/or dechlorinators are not chemically activeonce the microbial compositions are mixed with food and/or water to beadministered to the animal. In one embodiment, the one or more oxygenscavengers, denitrifiers, nitrifiers, heavy metal chelators, and/ordechlorinators are not chemically active when administered to theanimal.

In some embodiments, microbial compositions of the present disclosureoccur in a solid form (e.g., dispersed lyophilized spores) or a liquidform (microbes interspersed in a storage medium). In some embodiments,microbial compositions of the present disclosure are added in dry formto a liquid to a liquid to form a suspension immediately prior toadministration

In some embodiments, microbial compositions of the present disclosurecomprise one or more preservatives. The preservatives may be in liquidor gas formulations. The preservatives may be selected from one or moreof monosaccharide, disaccharide, trisaccharide, polysaccharide, aceticacid, ascorbic acid, calcium ascorbate, erythorbic acid, iso-ascorbicacid, erythrobic acid, potassium nitrate, sodium ascorbate, sodiumerythorbate, sodium iso-ascorbate, sodium nitrate, sodium nitrite,nitrogen, benzoic acid, calcium sorbate, ethyl lauroyl arginate,methyl-p-hydroxy benzoate, methyl paraben, potassium acetate, potassiumbenzoiate, potassium bisulphite, potassium diacetate, potassium lactate,potassium metabisulphite, potassium sorbate, propyl-p-hydroxy benzoate,propyl paraben, sodium acetate, sodium benzoate, sodium bisulphite,sodium nitrite, sodium diacetate, sodium lactate, sodium metabisulphite,sodium salt of methyl-p-hydroxy benzoic acid, sodium salt ofpropyl-p-hydroxy benzoic acid, sodium sulphate, sodium sulfite, sodiumdithionite, sulphurous acid, calcium propionate, dimethyl dicarbonate,natamycin, potassium sorbate, potassium bisulfite, potassiummetabisulfite, propionic acid, sodium diacetate, sodium propionate,sodium sorbate, sorbic acid, ascorbic acid, ascorbyl palmitate, ascorbylstearate, butylated hydro-xyanisole, butylated hydroxytoluene (BHT),butylated hydroxyl anisole (BHA), citric acid, citric acid esters ofmono- and/or diglycerides, L-cysteine, L-cysteine hydrochloride, gumguaiacum, gum guaiac, lecithin, lecithin citrate, monoglyceride citrate,monoisopropyl citrate, propyl gallate, sodium metabisulphite, tartaricacid, tertiary butyl hydroquinone, stannous chloride, thiodipropionicacid, dilauryl thiodipropionate, distearyl thiodipropionate, ethoxyquin,sulfur dioxide, formic acid, or tocopherol(s).

In some embodiments, microbial compositions of the present disclosurecomprise one or more oxygen scavengers, denitrifiers, nitrifiers, heavymetal chelators, and/or dechlorinators; and combinations thereof. In oneembodiment, the one or more oxygen scavengers, denitrifiers, nitrifiers,heavy metal chelators, and/or dechlorinators are not chemically activeonce the microbial compositions are mixed with food and/or water to beadministered to the beef cattle. In one embodiment, the one or moreoxygen scavengers, denitrifiers, nitrifiers, heavy metal chelators,and/or dechlorinators are not chemically active when administered to thebeef cattle.

In some embodiments, microbial compositions of the present disclosureinclude bacterial and/or fungal cells in spore form, vegetative cellform, and/or lysed cell form. In one embodiment, the lysed cell formacts as a mycotoxin binder, e.g. mycotoxins binding to dead cells.

In some embodiments, the microbial compositions are shelf stable in arefrigerator (35-40° F.) for a period of at least 1, 2, 3, 4, 5, 6, 7,8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25,26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43,44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, or 60days. In some embodiments, the microbial compositions are shelf stablein a refrigerator (35-40° F.) for a period of at least 1, 2, 3, 4, 5, 6,7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25,26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43,44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, or 60weeks.

In some embodiments, the microbial compositions are shelf stable at roomtemperature (68-72° F.) or between 50-77° F. fora period of at least 1,2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21,22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39,40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57,58, 59, or 60 days. In some embodiments, the microbial compositions areshelf stable at room temperature (68-72° F.) or between 50-77° F. for aperiod of at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15,16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33,34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51,52, 53, 54, 55, 56, 57, 58, 59, or 60 weeks.

In some embodiments, the microbial compositions are shelf stable at−23-35° F. for a period of at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11,12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29,30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47,48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, or 60 days. In someembodiments, the microbial compositions are shelf stable at −23-35° F.for a period of at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14,15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32,33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50,51, 52, 53, 54, 55, 56, 57, 58, 59, or 60 weeks.

In some embodiments, the microbial compositions are shelf stable at77-100° F. for a period of at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11,12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29,30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47,48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, or 60 days. In someembodiments, the microbial compositions are shelf stable at 77-100° F.for a period of at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14,15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32,33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50,51, 52, 53, 54, 55, 56, 57, 58, 59, or 60 weeks.

In some embodiments, the microbial compositions are shelf stable at101-213° F. for a period of at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11,12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29,30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47,48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, or 60 days. In someembodiments, the microbial compositions are shelf stable at 101-213° F.for a period of at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14,15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32,33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50,51, 52, 53, 54, 55, 56, 57, 58, 59, or 60 weeks.

In some embodiments, the microbial compositions of the presentdisclosure are shelf stable at refrigeration temperatures (35-40° F.),at room temperature (68-72° F.), between 50-77° F., between −23-35° F.,between 70-100° F., or between 101-213° F. fora period of about 1 to100, about 1 to 95, about 1 to 90, about 1 to 85, about 1 to 80, about 1to 75, about 1 to 70, about 1 to 65, about 1 to 60, about 1 to 55, about1 to 50, about 1 to 45, about 1 to 40, about 1 to 35, about 1 to 30,about 1 to 25, about 1 to 20, about 1 to 15, about 1 to 10, about 1 to5, about 5 to 100, about 5 to 95, about 5 to 90, about 5 to 85, about 5to 80, about 5 to 75, about 5 to 70, about 5 to 65, about 5 to 60, about5 to 55, about 5 to 50, about 5 to 45, about 5 to 40, about 5 to 35,about 5 to 30, about 5 to 25, about 5 to 20, about 5 to 15, about 5 to10, about 10 to 100, about 10 to 95, about 10 to 90, about 10 to 85,about 10 to 80, about 10 to 75, about 10 to 70, about 10 to 65, about 10to 60, about 10 to 55, about 10 to 50, about 10 to 45, about 10 to 40,about 10 to 35, about 10 to 30, about 10 to 25, about 10 to 20, about 10to 15, about 15 to 100, about 15 to 95, about 15 to 90, about 15 to 85,about 15 to 80, about 15 to 75, about 15 to 70, about 15 to 65, about 15to 60, about 15 to 55, about 15 to 50, about 15 to 45, about 15 to 40,about 15 to 35, about 15 to 30, about 15 to 25, about 15 to 20, about 20to 100, about 20 to 95, about 20 to 90, about 20 to 85, about 20 to 80,about 20 to 75, about 20 to 70, about 20 to 65, about 20 to 60, about 20to 55, about 20 to 50, about 20 to 45, about 20 to 40, about 20 to 35,about 20 to 30, about 20 to 25, about 25 to 100, about 25 to 95, about25 to 90, about 25 to 85, about 25 to 80, about 25 to 75, about 25 to70, about 25 to 65, about 25 to 60, about 25 to 55, about 25 to 50,about 25 to 45, about 25 to 40, about 25 to 35, about 25 to 30, about 30to 100, about 30 to 95, about 30 to 90, about 30 to 85, about 30 to 80,about 30 to 75, about 30 to 70, about 30 to 65, about 30 to 60, about 30to 55, about 30 to 50, about 30 to 45, about 30 to 40, about 30 to 35,about 35 to 100, about 35 to 95, about 35 to 90, about 35 to 85, about35 to 80, about 35 to 75, about 35 to 70, about 35 to 65, about 35 to60, about 35 to 55, about 35 to 50, about 35 to 45, about 35 to 40,about 40 to 100, about 40 to 95, about 40 to 90, about 40 to 85, about40 to 80, about 40 to 75, about 40 to 70, about 40 to 65, about 40 to60, about 40 to 55, about 40 to 50, about 40 to 45, about 45 to 100,about 45 to 95, about 45 to 90, about 45 to 85, about 45 to 80, about 45to 75, about 45 to 70, about 45 to 65, about 45 to 60, about 45 to 55,about 45 to 50, about 50 to 100, about 50 to 95, about 50 to 90, about50 to 85, about 50 to 80, about 50 to 75, about 50 to 70, about 50 to65, about 50 to 60, about 50 to 55, about 55 to 100, about 55 to 95,about 55 to 90, about 55 to 85, about 55 to 80, about 55 to 75, about 55to 70, about 55 to 65, about 55 to 60, about 60 to 100, about 60 to 95,about 60 to 90, about 60 to 85, about 60 to 80, about 60 to 75, about 60to 70, about 60 to 65, about 65 to 100, about 65 to 95, about 65 to 90,about 65 to 85, about 65 to 80, about 65 to 75, about 65 to 70, about 70to 100, about 70 to 95, about 70 to 90, about 70 to 85, about 70 to 80,about 70 to 75, about 75 to 100, about 75 to 95, about 75 to 90, about75 to 85, about 75 to 80, about 80 to 100, about 80 to 95, about 80 to90, about 80 to 85, about 85 to 100, about 85 to 95, about 85 to 90,about 90 to 100, about 90 to 95, or 95 to 100 weeks

In some embodiments, the microbial compositions of the presentdisclosure are shelf stable at refrigeration temperatures (35-40° F.),at room temperature (68-72° F.), between 50-77° F., between −23-35° F.,between 70-100° F., or between 101-213° F. for a period of 1 to 100, 1to 95, 1 to 90, 1 to 85, 1 to 80, 1 to 75, 1 to 70, 1 to 65, 1 to 60, 1to 55, 1 to 50, 1 to 45, 1 to 40, 1 to 35, 1 to 30, 1 to 25, 1 to 20, 1to 15, 1 to 10, 1 to 5, 5 to 100, 5 to 95, 5 to 90, 5 to 85, 5 to 80, 5to 75, 5 to 70, 5 to 65, 5 to 60, 5 to 55, 5 to 50, 5 to 45, 5 to 40, 5to 35, 5 to 30, 5 to 25, 5 to 20, 5 to 15, 5 to 10, 10 to 100, 10 to 95,10 to 90, 10 to 85, 10 to 80, 10 to 75, 10 to 70, 10 to 65, 10 to 60, 10to 55, 10 to 50, 10 to 45, 10 to 40, 10 to 35, 10 to 30, 10 to 25, 10 to20, 10 to 15, 15 to 100, 15 to 95, 15 to 90, 15 to 85, 15 to 80, 15 to75, 15 to 70, 15 to 65, 15 to 60, 15 to 55, 15 to 50, 15 to 45, 15 to40, 15 to 35, 15 to 30, 15 to 25, 15 to 20, 20 to 100, 20 to 95, 20 to90, 20 to 85, 20 to 80, 20 to 75, 20 to 70, 20 to 65, 20 to 60, 20 to55, 20 to 50, 20 to 45, 20 to 40, 20 to 35, 20 to 30, 20 to 25, 25 to100, 25 to 95, 25 to 90, 25 to 85, 25 to 80, 25 to 75, 25 to 70, 25 to65, 25 to 60, 25 to 55, 25 to 50, 25 to 45, 25 to 40, 25 to 35, 25 to30, 30 to 100, 30 to 95, 30 to 90, 30 to 85, 30 to 80, 30 to 75, 30 to70, 30 to 65, 30 to 60, 30 to 55, 30 to 50, 30 to 45, 30 to 40, 30 to35, 35 to 100, 35 to 95, 35 to 90, 35 to 85, 35 to 80, 35 to 75, 35 to70, 35 to 65, 35 to 60, 35 to 55, 35 to 50, 35 to 45, 35 to 40, 40 to100, 40 to 95, 40 to 90, 40 to 85, 40 to 80, 40 to 75, 40 to 70, 40 to65, 40 to 60, 40 to 55, 40 to 50, 40 to 45, 45 to 100, 45 to 95, 45 to90, 45 to 85, 45 to 80, 45 to 75, 45 to 70, 45 to 65, 45 to 60, 45 to55, 45 to 50, 50 to 100, 50 to 95, 50 to 90, 50 to 85, 50 to 80, 50 to75, 50 to 70, 50 to 65, 50 to 60, 50 to 55, 55 to 100, 55 to 95, 55 to90, 55 to 85, 55 to 80, 55 to 75, 55 to 70, 55 to 65, 55 to 60, 60 to100, 60 to 95, 60 to 90, 60 to 85, 60 to 80, 60 to 75, 60 to 70, 60 to65, 65 to 100, 65 to 95, 65 to 90, 65 to 85, 65 to 80, 65 to 75, 65 to70, 70 to 100, 70 to 95, 70 to 90, 70 to 85, 70 to 80, 70 to 75, 75 to100, 75 to 95, 75 to 90, 75 to 85, 75 to 80, 80 to 100, 80 to 95, 80 to90, 80 to 85, 85 to 100, 85 to 95, 85 to 90, 90 to 100, 90 to 95, or 95to 100 weeks.

In some embodiments, the microbial compositions of the presentdisclosure are shelf stable at refrigeration temperatures (35-40° F.),at room temperature (68-72° F.), between 50-77° F., between −23-35° F.,between 70-100° F., or between 101-213° F. for a period of about 1 to36, about 1 to 34, about 1 to 32, about 1 to 30, about 1 to 28, about 1to 26, about 1 to 24, about 1 to 22, about 1 to 20, about 1 to 18, about1 to 16, about 1 to 14, about 1 to 12, about 1 to 10, about 1 to 8,about 1 to 6, about 1 one 4, about 1 to 2, about 4 to 36, about 4 to 34,about 4 to 32, about 4 to 30, about 4 to 28, about 4 to 26, about 4 to24, about 4 to 22, about 4 to 20, about 4 to 18, about 4 to 16, about 4to 14, about 4 to 12, about 4 to 10, about 4 to 8, about 4 to 6, about 6to 36, about 6 to 34, about 6 to 32, about 6 to 30, about 6 to 28, about6 to 26, about 6 to 24, about 6 to 22, about 6 to 20, about 6 to 18,about 6 to 16, about 6 to 14, about 6 to 12, about 6 to 10, about 6 to8, about 8 to 36, about 8 to 34, about 8 to 32, about 8 to 30, about 8to 28, about 8 to 26, about 8 to 24, about 8 to 22, about 8 to 20, about8 to 18, about 8 to 16, about 8 to 14, about 8 to 12, about 8 to 10,about 10 to 36, about 10 to 34, about 10 to 32, about 10 to 30, about 10to 28, about 10 to 26, about 10 to 24, about 10 to 22, about 10 to 20,about 10 to 18, about 10 to 16, about 10 to 14, about 10 to 12, about 12to 36, about 12 to 34, about 12 to 32, about 12 to 30, about 12 to 28,about 12 to 26, about 12 to 24, about 12 to 22, about 12 to 20, about 12to 18, about 12 to 16, about 12 to 14, about 14 to 36, about 14 to 34,about 14 to 32, about 14 to 30, about 14 to 28, about 14 to 26, about 14to 24, about 14 to 22, about 14 to 20, about 14 to 18, about 14 to 16,about 16 to 36, about 16 to 34, about 16 to 32, about 16 to 30, about 16to 28, about 16 to 26, about 16 to 24, about 16 to 22, about 16 to 20,about 16 to 18, about 18 to 36, about 18 to 34, about 18 to 32, about 18to 30, about 18 to 28, about 18 to 26, about 18 to 24, about 18 to 22,about 18 to 20, about 20 to 36, about 20 to 34, about 20 to 32, about 20to 30, about 20 to 28, about 20 to 26, about 20 to 24, about 20 to 22,about 22 to 36, about 22 to 34, about 22 to 32, about 22 to 30, about 22to 28, about 22 to 26, about 22 to 24, about 24 to 36, about 24 to 34,about 24 to 32, about 24 to 30, about 24 to 28, about 24 to 26, about 26to 36, about 26 to 34, about 26 to 32, about 26 to 30, about 26 to 28,about 28 to 36, about 28 to 34, about 28 to 32, about 28 to 30, about 30to 36, about 30 to 34, about 30 to 32, about 32 to 36, about 32 to 34,or about 34 to 36 months.

In some embodiments, the microbial compositions of the presentdisclosure are shelf stable at refrigeration temperatures (35-40° F.),at room temperature (68-72° F.), between 50-77° F., between −23-35° F.,between 70-100° F., or between 101-213° F. for a period of 1 to 36 1 to34 1 to 32 1 to 30 1 to 28 1 to 26 1 to 24 1 to 22 1 to 20 1 to 18 1 to16 1 to 14 1 to 12 1 to 10 1 to 8 1 to 6 1 one 4 1 to 24 to 36 4 to 34 4to 32 4 to 30 4 to 28 4 to 26 4 to 24 4 to 22 4 to 20 4 to 18 4 to 16 4to 14 4 to 12 4 to 10 4 to 8 4 to 6 6 to 36 6 to 34 6 to 32 6 to 30 6 to28 6 to 26 6 to 24 6 to 22 6 to 20 6 to 18 6 to 16 6 to 14 6 to 12 6 to10 6 to 8 8 to 36 8 to 34 8 to 32 8 to 30 8 to 28 8 to 26 8 to 24 8 to22 8 to 20 8 to 18 8 to 16 8 to 14 8 to 12 8 to 10 10 to 36 10 to 34 10to 32 10 to 30 10 to 28 10 to 26 10 to 24 10 to 22 10 to 20 10 to 18 10to 16 10 to 14 10 to 12 12 to 36 12 to 34 12 to 32 12 to 30 12 to 28 12to 26 12 to 24 12 to 22 12 to 20 12 to 18 12 to 16 12 to 14 14 to 36 14to 34 14 to 32 14 to 30 14 to 28 14 to 26 14 to 24 14 to 22 14 to 20 14to 18 14 to 16 16 to 36 16 to 34 16 to 32 16 to 30 16 to 28 16 to 26 16to 24 16 to 22 16 to 20 16 to 18 18 to 36 18 to 34 18 to 32 18 to 30 18to 28 18 to 26 18 to 24 18 to 22 18 to 20 20 to 36 20 to 34 20 to 32 20to 30 20 to 28 20 to 26 20 to 24 20 to 22 22 to 36 22 to 34 22 to 32 22to 30 22 to 28 22 to 26 22 to 24 24 to 36 24 to 34 24 to 32 24 to 30 24to 28 24 to 26 26 to 36 26 to 34 26 to 32 26 to 30 26 to 28 28 to 36 28to 34 28 to 32 28 to 30 30 to 36 30 to 34 30 to 32 32 to 36 32 to 34, orabout 34 to 36.

In some embodiments, the microbial compositions of the presentdisclosure are shelf stable at any of the disclosed temperatures and/ortemperature ranges and spans of time at a relative humidity of at least1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20,21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38,39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56,57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74,75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92,93, 94, 95, 96, 97, or 98%.

In some embodiments, the microbial composition of the present disclosurepossesses a water activity (aw) of less than 0.750, 0.700, 0.650, 0.600,0.550, 0.500, 0.475, 0.450, 0.425, 0.400, 0.375, 0.350, 0.325, 0.300,0.275, 0.250, 0.225, 0.200, 0.190, 0.180, 0.170, 0.160, 0.150, 0.140,0.130, 0.120, 0.110, 0.100, 0.095, 0.090, 0.085, 0.080, 0.075, 0.070,0.065, 0.060, 0.055, 0.050, 0.045, 0.040, 0.035, 0.030, 0.025, 0.020,0.015, 0.010, or 0.005.

In some embodiments, the microbial composition of the present disclosurepossesses a water activity (aw) of less than about 0.750, about 0.700,about 0.650, about 0.600, about 0.550, about 0.500, about 0.475, about0.450, about 0.425, about 0.400, about 0.375, about 0.350, about 0.325,about 0.300, about 0.275, about 0.250, about 0.225, about 0.200, about0.190, about 0.180, about 0.170, about 0.160, about 0.150, about 0.140,about 0.130, about 0.120, about 0.110, about 0.100, about 0.095, about0.090, about 0.085, about 0.080, about 0.075, about 0.070, about 0.065,about 0.060, about 0.055, about 0.050, about 0.045, about 0.040, about0.035, about 0.030, about 0.025, about 0.020, about 0.015, about 0.010,or about 0.005.

The water activity values are determined by the method of SaturatedAqueous Solutions (Multon, “Techniques d'Analyse E De Controle Dans LesIndustries Agroalimentaires” APRIA (1981)) or by direct measurementusing a viable Robotronic BT hygrometer or other hygrometer orhygroscope.

In some embodiments, the microbial composition comprises at least twodifferent microbes, and wherein the at least two microbes are present inthe composition at a ratio of 1:2, 1:3, 1:3, 1:5, 1:6, 1:7, 1:8, 1:9,1:10, 1:11, 1:12, 1:13, 1:14, 1:15, 1:16, 1:17, 1:18, 1:19, 1:20, 1:21,1:22, 1:23, 1:24, 1:25, 1:26, 1:27, 1:28, 1:29, 1:30, 1:40, 1:50, 1:60,1:100, 1:125, 1:150, 1:175, or 1:200 or the inverse thereof. In someembodiments, the microbial composition comprises at least threedifferent microbes, and wherein the three microbes are present in thecomposition at a ratio of 1:2:1, 1:1:2, 2:2:1, 1:3:1, 1:1:3, 3:1:1,3:3:1, 1:5:1, 1:1:5, 5:1:1, 5:5:1, or 1:5:5.

Encapsulation Compositions

In some embodiments, the microbes or microbial compositions of thedisclosure are encapsulated in an encapsulating composition. Anencapsulating composition protects the microbes from external stressorsprior to entering the gastrointestinal tract of beef cattle. In someembodiments, external stressors include thermal and physical stressorsassociated with pelleting and extrusion. In some embodiments, externalstressors include chemicals present in the compositions. Encapsulatingcompositions further create an environment that may be beneficial to themicrobes, such as minimizing the oxidative stresses of an aerobicenvironment on anaerobic microbes. See Kalsta et al. (U.S. Pat. No.5,104,662A), Ford (U.S. Pat. No. 5,733,568A), and Mosbach and Nilsson(U.S. Pat. No. 4,647,536A) for encapsulation compositions of microbes,and methods of encapsulating microbes.

In one embodiment, the compositions of the present disclosure exhibit athermal tolerance, which is used interchangeably with heat tolerance andheat resistance. In one embodiment, thermal tolerant compositions of thepresent disclosure are tolerant of the high temperatures associated withfeed manufacturing, mixing of feed and compositions of the presentdisclosure, storage in high heat environments, etc. In one embodiment,thermal tolerant compositions of the present disclosure are resistant toheat-killing and denaturation of the cell wall components and theintracellular environment.

In one embodiments, the encapsulation is a reservoir-type encapsulation.In one embodiment, the encapsulation is a matrix-type encapsulation. Inone embodiment, the encapsulation is a coated matrix-type encapsulation.Burgain et al. (2011. J. Food Eng. 104:467-483) discloses numerousencapsulation embodiments and techniques, all of which are incorporatedby reference.

In some embodiments, the compositions of the present disclosure areencapsulated in one or more of the following: gellan gum, xanthan gum,K-Carrageenan, cellulose acetate phthalate, chitosan, starch, milk fat,whey protein, Ca-alginate, raftilose, raftiline, pectin, saccharide,glucose, maltodextrin, gum arabic, guar, seed flour, alginate, dextrins,dextrans, celluloase, gelatin, gelatin, albumin, casein, gluten, acaciagum, tragacanth, wax, paraffin, stearic acid, monodiglycerides, anddiglycerides. In some embodiments, the compositions of the presentdisclosure are encapsulated by one or more of a polymer, carbohydrate,sugar, plastic, glass, polysaccharide, lipid, wax, oil, fatty acid, orglyceride. In one embodiment, the microbial composition is encapsulatedby glucose. In one embodiment, the microbial composition is encapsulatedby a glucose-containing composition. In one embodiment, formulations ofthe microbial composition comprise a glucose encapsulant. In oneembodiment, formulations of the microbial composition comprise aglucose-encapsulated composition.

In some embodiments, the encapsulation of the compositions of thepresent disclosure is carried out by an extrusion, emulsification,coating, agglomeration, lyophilization, vitrification, foam drying,preservation by vaporization, vacuum-drying, or spray-drying.

In some embodiments, the encapsulated compositions of the presentdisclosure are vitrified. In some embodiments, encapsulation involves aprocess of drying a composition of the present disclosure in thepresence of a substance which forms a glassy, amorphous solid state, aprocess known as vitrification, and in doing so encapsulates thecomposition. In some embodiments, the vitrified composition is protectedfrom degradative conditions that would typically destroy or degrademicrobes. Many common substances have the property of vitrification;that is, they will form a glassy solid state under certain conditions.Among these substances are several sugars, including sucrose andmaltose, and other more complex compounds, such as polyvinyl pyrolidone(PVP). As any solution dries down, the molecules in the solution caneither crystalize, or they can vitrify. A solute which has an extensiveasymmetry may be a superior vitrifier, because of the hindrances tonucleation of crystals during drying. A substance that inhibits thecrystallization of another substance may result in the combinedsubstances forming a superior vitrification, such as raffinose in thepresence of sucrose. See U.S. Pat. Nos. 5,290,765 and 9,469,835.

In some embodiments, a microbial composition is produced that isencapsulated in a vitrified substance. The vitrified composition may becreated by selecting a mixture including cells; combining said mixturewith sufficient quantity of one or more vitrifying solutes to protectsaid mixture during drying and to inhibit destructive reactions; anddrying said combination by exposing said combination to a desiccant, ordesiccating conditions, at a temperature above that which saidcombination will freeze and below that at which said vitrifying solutesachieve the vitrified state, at approximately normal atmosphericpressure, until said combination is substantially dry.

In one embodiment, the encapsulating composition comprises microcapsuleshaving a multiplicity of liquid cores encapsulated in a solid shellmaterial. For purposes of the disclosure, a “multiplicity” of cores isdefined as two or more.

A first category of useful fusible shell materials is that of normallysolid fats, including fats which are already of suitable hardness andanimal or vegetable fats and oils which are hydrogenated until theirmelting points are sufficiently high to serve the purposes of thepresent disclosure. Depending on the desired process and storagetemperatures and the specific material selected, a particular fat can beeither a normally solid or normally liquid material. The terms “normallysolid” and “normally liquid” as used herein refer to the state of amaterial at desired temperatures for storing the resultingmicrocapsules. Since fats and hydrogenated oils do not, strictlyspeaking, have melting points, the term “melting point” is used hereinto describe the minimum temperature at which the fusible materialbecomes sufficiently softened or liquid to be successfully emulsifiedand spray cooled, thus roughly corresponding to the maximum temperatureat which the shell material has sufficient integrity to prevent releaseof the choline cores. “Melting point” is similarly defined herein forother materials which do not have a sharp melting point.

Specific examples of fats and oils useful herein (some of which requirehardening) are as follows: animal oils and fats, such as beef tallow,mutton tallow, lamb tallow, lard or pork fat, fish oil, and sperm oil;vegetable oils, such as canola oil, cottonseed oil, peanut oil, cornoil, olive oil, soybean oil, sunflower oil, safflower oil, coconut oil,palm oil, linseed oil, tung oil, and castor oil; fatty acidmonoglycerides and diglycerides; free fatty acids, such as stearic acid,palmitic acid, and oleic acid; and mixtures thereof. The above listingof oils and fats is not meant to be exhaustive, but only exemplary.

Specific examples of fatty acids include linoleic acid, γ-linoleic acid,dihomo-γ-linolenic acid, arachidonic acid, docosatetraenoic acid,vaccenic acid, nervonic acid, mead acid, erucic acid, gondoic acid,elaidic acid, oleic acid, palitoleic acid, stearidonic acid,eicosapentaenoic acid, valeric acid, caproic acid, enanthic acid,caprylic acid, pelargonic acid, capric acid, undecylic acid, lauricacid, tridecylic acid, myristic acid, pentadecylic acid, palmitic acid,margaric acid, stearic acid, nonadecyclic acid, arachidic acid,heneicosylic acid, behenic acid, tricosylic acid, lignoceric acid,pentacosylic acid, cerotic acid, heptacosylic acid, montanic acid,nonacosylic acid, melissic acid, henatriacontylic acid, lacceroic acid,psyllic acid, geddic acid, ceroplastic acid, hexatriacontylic acid,heptatriacontanoic acid, and octatriacontanoic acid.

Another category of fusible materials useful as encapsulating shellmaterials is that of waxes. Representative waxes contemplated for useherein are as follows: animal waxes, such as beeswax, lanolin, shellwax, and Chinese insect wax; vegetable waxes, such as carnauba,candelilla, bayberry, and sugar cane; mineral waxes, such as paraffin,microcrystalline petroleum, ozocerite, ceresin, and montan; syntheticwaxes, such as low molecular weight polyolefin (e.g., CARBOWAX), andpolyol ether-esters (e.g., sorbitol); Fischer-Tropsch process syntheticwaxes; and mixtures thereof. Water-soluble waxes, such as CARBOWAX andsorbitol, are not contemplated herein if the core is aqueous.

Still other fusible compounds useful herein are fusible natural resins,such as rosin, balsam, shellac, and mixtures thereof.

In some embodiments, the microbes or microbial composition is embeddedin a wax, such as the waxes described in the present disclosure.

In some embodiments, the microbes or microbial composition is embeddedin wax balls. In some embodiments, the microbes or microbial compositionis already encapsulated prior to being embedded in wax balls. In someembodiments, the wax balls are 10 microbes, 20 microns, 30 microns, 40microns, 50 microns, 60 microns, 70 microns, 80 microns, 90 microns, 100microns, 150 microns, 200 microns, 250 microns, 300 microns, 350microns, 400 microns, 450 microns, 500 microns, 550 microns, 600microns, 650 microns, 700 microns, 750 microns, 800 microns, 850microns, 900 microns, 950 microns, or 1,000 microns.

In some embodiments, the wax balls are about 10 microbes, about 20microns, about 30 microns, about 40 microns, about 50 microns, about 60microns, about 70 microns, about 80 microns, about 90 microns, about 100microns, about 150 microns, about 200 microns, about 250 microns, about300 microns, about 350 microns, about 400 microns, about 450 microns,about 500 microns, about 550 microns, about 600 microns, about 650microns, about 700 microns, about 750 microns, about 800 microns, about850 microns, about 900 microns, about 950 microns, or about 1,000microns.

In some embodiments, the wax balls are between 10-20 microns, 10-30microns, 10-40 microns, 10-50 microns, 10-60 microns, 10-70 microns,10-80 microns, 10-90 microns, 10-100 microns, 10-250 microns, 10-500microns, 10-750 microns, 10-1,000 microns, 20-30 microns, 20-40 microns,20-50 microns, 20-60 microns, 20-70 microns, 20-80 microns, 20-90microns, 20-100 microns, 20-250 microns, 20-500 microns, 20-750 microns,20-1,000 microns, 30-40 microns, 30-50 microns, 30-60 microns, 30-70microns, 30-80 microns, 30-90 microns, 30-100 microns, 30-250 microns,30-500 microns, 30-750 microns, 30-1,000 microns, 40-50 microns, 40-60microns, 40-70 microns, 40-80 microns, 40-90 microns, 40-100 microns,40-250 microns, 40-500 microns, 40-750 microns, 40-1,000 microns, 50-60microns, 50-70 microns, 50-80 microns, 50-90 microns, 50-100 microns,50-250 microns, 50-500 microns, 50-750 microns, 50-1,000 microns, 60-70microns, 60-80 microns, 60-90 microns, 60-100 microns, 60-250 microns,60-500 microns, 60-750 microns, 60-1,000 microns, 70-80 microns 70-90microns, 70-90 microns, 70-100 microns, 70-250 microns, 70-500 microns,70-750 microns, 70-1,000 microns, 80-90 microns, 80-100 microns, 80-250microns, 80-500 microns, 80-500 microns, 80-750 microns, 80-1,000microns, 90-100 microns, 90-250 microns, 90-500 microns, 90-750 microns,90-1,000 microns, 100-250 microns, 100-500 microns, 100-750 microns,100-1,000 microns, 250-500 microns, 250-750 microns, 250-1,000 microns,500-750 microns, 500-1,000 microns, or 750-1,000 microns.

In some embodiments, the wax balls are between about 10-20 microns,about 10-30 microns, about 10-40 microns, about 10-50 microns, about10-60 microns, about 10-70 microns, about 10-80 microns, about 10-90microns, about 10-100 microns, about 10-250 microns, about 10-500microns, about 10-750 microns, about 10-1,000 microns, about 20-30microns, about 20-40 microns, about 20-50 microns, about 20-60 microns,about 20-70 microns, about 20-80 microns, about 20-90 microns, about20-100 microns, about 20-250 microns, about 20-500 microns, about 20-750microns, about 20-1,000 microns, about 30-40 microns, about 30-50microns, about 30-60 microns, about 30-70 microns, about 30-80 microns,about 30-90 microns, about 30-100 microns, about 30-250 microns, about30-500 microns, about 30-750 microns, about 30-1,000 microns, about40-50 microns, about 40-60 microns, about 40-70 microns, about 40-80microns, about 40-90 microns, about 40-100 microns, about 40-250microns, about 40-500 microns, about 40-750 microns, about 40-1,000microns, about 50-60 microns, about 50-70 microns, about 50-80 microns,about 50-90 microns, about 50-100 microns, about 50-250 microns, about50-500 microns, about 50-750 microns, about 50-1,000 microns, about60-70 microns, about 60-80 microns, about 60-90 microns, about 60-100microns, about 60-250 microns, about 60-500 microns, about 60-750microns, about 60-1,000 microns, about 70-80 microns about 70-90microns, about 70-90 microns, about 70-100 microns, about 70-250microns, about 70-500 microns, about 70-750 microns, about 70-1,000microns, about 80-90 microns, about 80-100 microns, about 80-250microns, about 80-500 microns, about 80-500 microns, about 80-750microns, about 80-1,000 microns, about 90-100 microns, about 90-250microns, about 90-500 microns, about 90-750 microns, about 90-1,000microns, about 100-250 microns, about 100-500 microns, about 100-750microns, about 100-1,000 microns, about 250-500 microns, about 250-750microns, about 250-1,000 microns, about 500-750 microns, about 500-1,000microns, or about 750-1,000 microns.

Various adjunct materials are contemplated for incorporation in fusiblematerials according to the present disclosure. For example,antioxidants, light stabilizers, dyes and lakes, flavors, essentialoils, anti-caking agents, fillers, pH stabilizers, sugars(monosaccharides, disaccharides, trisaccharides, and polysaccharides)and the like can be incorporated in the fusible material in amountswhich do not diminish its utility for the present disclosure.

The core material contemplated herein constitutes from about 0.1% toabout 50%, about 1% to about 35%. or about 5% to about 30% by weight ofthe microcapsules. In some embodiments, the core material contemplatedherein constitutes no more than about 30% by weight of themicrocapsules. In some embodiments, the core material contemplatedherein constitutes about 5% by weight of the microcapsules. The corematerial is contemplated as either a liquid or solid at contemplatedstorage temperatures of the microcapsules.

The cores may include other additives well-known in the pharmaceuticalart, including edible sugars, such as sucrose, glucose, maltose,fructose, lactose, cellobiose, monosaccharides, disaccharides,trisaccharides, and polysaccharides, and mixtures thereof; artificialsweeteners, such as aspartame, saccharin, cyclamate salts, and mixturesthereof; edible acids, such as acetic acid (vinegar), citric acid,ascorbic acid, tartaric acid, and mixtures thereof; edible starches,such as corn starch; hydrolyzed vegetable protein; water-solublevitamins, such as Vitamin C; water-soluble medicaments; water-solublenutritional materials, such as ferrous sulfate; flavors; salts;monosodium glutamate; antimicrobial agents, such as sorbic acid;antimycotic agents, such as potassium sorbate, sorbic acid, sodiumbenzoate, and benzoic acid; food grade pigments and dyes; and mixturesthereof. Other potentially useful supplemental core materials will beapparent to those of ordinary skill in the art.

Emulsifying agents may be employed to assist in the formation of stableemulsions. Representative emulsifying agents include glycerylmonostearate, polysorbate esters, ethoxylated mono- and diglycerides,and mixtures thereof.

For ease of processing, and particularly to enable the successfulformation of a reasonably stable emulsion, the viscosities of the corematerial and the shell material should be similar at the temperature atwhich the emulsion is formed. In particular, the ratio of the viscosityof the shell to the viscosity of the core, expressed in centipoise orcomparable units, and both measured at the temperature of the emulsion,should be from about 22:1 to about 1:1, desirably from about 8:1 toabout 1:1, and preferably from about 3:1 to about 1:1. A ratio of 1:1would be ideal, but a viscosity ratio within the recited ranges isuseful.

Encapsulating compositions are not limited to microcapsule compositionsas disclosed above. In some embodiments encapsulating compositionsencapsulate the microbial compositions in an adhesive polymer that canbe natural or synthetic without toxic effect. In some embodiments, theencapsulating composition may be a matrix selected from sugar matrix,gelatin matrix, polymer matrix, silica matrix, starch matrix, foammatrix, glass/glassy matrix etc. See Pirzio et al. (U.S. Pat. No.7,488,503). In some embodiments, the encapsulating composition may beselected from polyvinyl acetates; polyvinyl acetate copolymers; ethylenevinyl acetate (EVA) copolymers; polyvinyl alcohols; polyvinyl alcoholcopolymers; celluloses, including ethylcelluloses, methylcelluloses,hydroxymethylcelluloses, hydroxypropylcelluloses andcarboxymethylcellulose; polyvinylpyrolidones; polysaccharides, includingstarch, modified starch, dextrins, maltodextrins, alginate andchitosans; monosaccharides; fats; fatty acids, including oils; proteins,including gelatin and zeins; gum arabics; shellacs; vinylidene chlorideand vinylidene chloride copolymers; calcium lignosulfonates; acryliccopolymers; polyvinylacrylates; polyethylene oxide; acrylamide polymersand copolymers; polyhydroxyethyl acrylate, methylacrylamide monomers;and polychloroprene.

In some embodiments, the encapsulating compositions comprise at leastone layer of encapsulation. In some embodiments, the encapsulatingcompositions comprise at least 1, at least 2, at least 3, at least 4, atleast 5, at least 6, at least 7, at least 8, at least 9, at least 10, atleast 11, at least 12, at least 13, at least 14, at least 15, at least16, at least 17, at least 18, at least 19, or at least 20 layers ofencapsulation/encapsulants.

In some embodiments, the encapsulating compositions comprise at leasttwo layers of encapsulation. In some embodiments, each layer ofencapsulation confers a different characteristic to the composition. Insome embodiments, no two consecutive layers confer the samecharacteristic. In some embodiments, at least one layer of the at leasttwo layers of encapsulation confers thermostability, shelf stability,ultraviolet resistance, moisture resistance, hydrophobicity,hydrophilicity, lipophobicity, lipophilicity, pH stability, acidresistance, and base resistance.

In some embodiments, the encapsulating compositions comprise two layersof encapsulation; the first layer confers thermostability and/or shelfstability, and the second layer provides pH resistance.

In some embodiments, the encapsulating layers confer a timed release ofthe microbial composition held in the center of the encapsulatinglayers. In some embodiments, the greater the number of layers confers agreater amount of time before the microbial composition is exposed, postadministration.

In some embodiments, the encapsulating shell of the present disclosurecan be up to 10 μm, 20 μm, 30 μm, 40 μm, 50 μm, 60 μm, 70 μm, 80 μm, 90μm, 100 μm, 110 μm, 120 μm, 130 μm, 140 μm, 150 μm, 160 μm, 170 μm, 180μm, 190 μm, 200 μm, 210 μm, 220 μm, 230 μm, 240 μm, 250 μm, 260 μm, 270μm, 280 μm, 290 μm, 300 μm, 310 μm, 320 μm, 330 μm, 340 μm, 350 μm, 360μm, 370 μm, 380 μm, 390 μm, 400 μm, 410 μm, 420 μm, 430 μm, 440 μm, 450μm, 460 μm, 470 μm, 480 μm, 490 μm, 500 μm, 510 μm, 520 μm, 530 μm, 540μm, 550 μm, 560 μm, 570 μm, 580 μm, 590 μm, 600 μm, 610 μm, 620 μm, 630μm, 640 μm, 650 μm, 660 μm, 670 μm, 680 μm, 690 μm, 700 μm, 710 μm, 720μm, 730 μm, 740 μm, 750 μm, 760 μm, 770 μm, 780 μm, 790 μm, 800 μm, 810μm, 820 μm, 830 μm, 840 μm, 850 μm, 860 μm, 870 μm, 880 μm, 890 μm, 900μm, 910 μm, 920 μm, 930 μm, 940 μm, 950 μm, 960 μm, 970 μm, 980 μm, 990μm, 1000 μm, 1010 μm, 1020 μm, 1030 μm, 1040 μm, 1050 μm, 1060 μm, 1070μm, 1080 μm, 1090 μm, 1100 μm, 1110 μm, 1120 μm, 1130 μm, 1140 μm, 1150μm, 1160 μm, 1170 μm, 1180 μm, 1190 μm, 1200 μm, 1210 μm, 1220 μm, 1230μm, 1240 μm, 1250 μm, 1260 μm, 1270 μm, 1280 μm, 1290 μm, 1300 μm, 1310μm, 1320 μm, 1330 μm, 1340 μm, 1350 μm, 1360 μm, 1370 μm, 1380 μm, 1390μm, 1400 μm, 1410 μm, 1420 μm, 1430 μm, 1440 μm, 1450 μm, 1460 μm, 1470μm, 1480 μm, 1490 μm, 1500 μm, 1510 μm, 1520 μm, 1530 μm, 1540 μm, 1550μm, 1560 μm, 1570 μm, 1580 μm, 1590 μm, 1600 μm, 1610 μm, 1620 μm, 1630μm, 1640 μm, 1650 μm, 1660 μm, 1670 μm, 1680 μm, 1690 μm, 1700 μm, 1710μm, 1720 μm, 1730 μm, 1740 μm, 1750 μm, 1760 μm, 1770 μm, 1780 μm, 1790μm, 1800 μm, 1810 μm, 1820 μm, 1830 μm, 1840 μm, 1850 μm, 1860 μm, 1870μm, 1880 μm, 1890 μm, 1900 μm, 1910 μm, 1920 μm, 1930 μm, 1940 μm, 1950μm, 1960 μm, 1970 μm, 1980 μm, 1990 μm, 2000 μm, 2010 μm, 2020 μm, 2030μm, 2040 μm, 2050 μm, 2060 μm, 2070 μm, 2080 μm, 2090 μm, 2100 μm, 2110μm, 2120 μm, 2130 μm, 2140 μm, 2150 μm, 2160 μm, 2170 μm, 2180 μm, 2190μm, 2200 μm, 2210 μm, 2220 μm, 2230 μm, 2240 μm, 2250 μm, 2260 μm, 2270μm, 2280 μm, 2290 μm, 2300 μm, 2310 μm, 2320 μm, 2330 μm, 2340 μm, 2350μm, 2360 μm, 2370 μm, 2380 μm, 2390 μm, 2400 μm, 2410 μm, 2420 μm, 2430μm, 2440 μm, 2450 μm, 2460 μm, 2470 μm, 2480 μm, 2490 μm, 2500 μm, 2510μm, 2520 μm, 2530 μm, 2540 μm, 2550 μm, 2560 μm, 2570 μm, 2580 μm, 2590μm, 2600 μm, 2610 μm, 2620 μm, 2630 μm, 2640 μm, 2650 μm, 2660 μm, 2670μm, 2680 μm, 2690 μm, 2700 μm, 2710 μm, 2720 μm, 2730 μm, 2740 μm, 2750μm, 2760 μm, 2770 μm, 2780 μm, 2790 μm, 2800 μm, 2810 μm, 2820 μm, 2830μm, 2840 μm, 2850 μm, 2860 μm, 2870 μm, 2880 μm, 2890 μm, 2900 μm, 2910μm, 2920 μm, 2930 μm, 2940 μm, 2950 μm, 2960 μm, 2970 μm, 2980 μm, 2990μm, or 3000 μm thick.

In some embodiments, the encapsulation composition of the presentdisclosure possesses a water activity (aw) of less than 0.750, 0.700,0.650, 0.600, 0.550, 0.500, 0.475, 0.450, 0.425, 0.400, 0.375, 0.350,0.325, 0.300, 0.275, 0.250, 0.225, 0.200, 0.190, 0.180, 0.170, 0.160,0.150, 0.140, 0.130, 0.120, 0.110, 0.100, 0.095, 0.090, 0.085, 0.080,0.075, 0.070, 0.065, 0.060, 0.055, 0.050, 0.045, 0.040, 0.035, 0.030,0.025, 0.020, 0.015, 0.010, or 0.005.

In some embodiments, the encapsulation composition of the presentdisclosure possesses a water activity (aw) of less than about 0.750,about 0.700, about 0.650, about 0.600, about 0.550, about 0.500, about0.475, about 0.450, about 0.425, about 0.400, about 0.375, about 0.350,about 0.325, about 0.300, about 0.275, about 0.250, about 0.225, about0.200, about 0.190, about 0.180, about 0.170, about 0.160, about 0.150,about 0.140, about 0.130, about 0.120, about 0.110, about 0.100, about0.095, about 0.090, about 0.085, about 0.080, about 0.075, about 0.070,about 0.065, about 0.060, about 0.055, about 0.050, about 0.045, about0.040, about 0.035, about 0.030, about 0.025, about 0.020, about 0.015,about 0.010, or about 0.005.

In one embodiment, the microbe(s) are first dried by spray dry,lyophilization, or foam drying along with excipients that may includeone or more sugars, sugar alcohols, disaccharides, trisaccharides,polysaccharides, salts, amino acids, amino acid salts, or polymers.

In some embodiments, the microbes or compositions comprising themicrobes are milled to a size of 10 microns, 20 microns, 30 microns, 40microns, 50 microns, 60 microns, 70 microns, 80 microns, 90 microns, 100microns, 150 microns, 200 microns, 250 microns, 300 microns, 350microns, 400 microns, 450 microns, 500 microns, 550 microns, 600microns, 650 microns, 700 microns, 750 microns, 800 microns, 850microns, 900 microns, 950 microns, or 1,000 microns.

In some embodiments, the microbes or compositions comprising themicrobes are milled to a size of about 10 microbes, about 20 microns,about 30 microns, about 40 microns, about 50 microns, about 60 microns,about 70 microns, about 80 microns, about 90 microns, about 100 microns,about 150 microns, about 200 microns, about 250 microns, about 300microns, about 350 microns, about 400 microns, about 450 microns, about500 microns, about 550 microns, about 600 microns, about 650 microns,about 700 microns, about 750 microns, about 800 microns, about 850microns, about 900 microns, about 950 microns, or about 1,000 microns.

In some embodiments, the microbes or compositions comprising themicrobes are milled to a size of between 10-20 microns, 10-30 microns,10-40 microns, 10-50 microns, 10-60 microns, 10-70 microns, 10-80microns, 10-90 microns, 10-100 microns, 10-250 microns, 10-500 microns,10-750 microns, 10-1,000 microns, 20-30 microns, 20-40 microns, 20-50microns, 20-60 microns, 20-70 microns, 20-80 microns, 20-90 microns,20-100 microns, 20-250 microns, 20-500 microns, 20-750 microns, 20-1,000microns, 30-40 microns, 30-50 microns, 30-60 microns, 30-70 microns,30-80 microns, 30-90 microns, 30-100 microns, 30-250 microns, 30-500microns, 30-750 microns, 30-1,000 microns, 40-50 microns, 40-60 microns,40-70 microns, 40-80 microns, 40-90 microns, 40-100 microns, 40-250microns, 40-500 microns, 40-750 microns, 40-1,000 microns, 50-60microns, 50-70 microns, 50-80 microns, 50-90 microns, 50-100 microns,50-250 microns, 50-500 microns, 50-750 microns, 50-1,000 microns, 60-70microns, 60-80 microns, 60-90 microns, 60-100 microns, 60-250 microns,60-500 microns, 60-750 microns, 60-1,000 microns, 70-80 microns 70-90microns, 70-90 microns, 70-100 microns, 70-250 microns, 70-500 microns,70-750 microns, 70-1,000 microns, 80-90 microns, 80-100 microns, 80-250microns, 80-500 microns, 80-500 microns, 80-750 microns, 80-1,000microns, 90-100 microns, 90-250 microns, 90-500 microns, 90-750 microns,90-1,000 microns, 100-250 microns, 100-500 microns, 100-750 microns,100-1,000 microns, 250-500 microns, 250-750 microns, 250-1,000 microns,500-750 microns, 500-1,000 microns, or 750-1,000 microns.

In some embodiments, the microbes or compositions comprising themicrobes are milled to a size of between about 10-20 microns, about10-30 microns, about 10-40 microns, about 10-50 microns, about 10-60microns, about 10-70 microns, about 10-80 microns, about 10-90 microns,about 10-100 microns, about 10-250 microns, about 10-500 microns, about10-750 microns, about 10-1,000 microns, about 20-30 microns, about 20-40microns, about 20-50 microns, about 20-60 microns, about 20-70 microns,about 20-80 microns, about 20-90 microns, about 20-100 microns, about20-250 microns, about 20-500 microns, about 20-750 microns, about20-1,000 microns, about 30-40 microns, about 30-50 microns, about 30-60microns, about 30-70 microns, about 30-80 microns, about 30-90 microns,about 30-100 microns, about 30-250 microns, about 30-500 microns, about30-750 microns, about 30-1,000 microns, about 40-50 microns, about 40-60microns, about 40-70 microns, about 40-80 microns, about 40-90 microns,about 40-100 microns, about 40-250 microns, about 40-500 microns, about40-750 microns, about 40-1,000 microns, about 50-60 microns, about 50-70microns, about 50-80 microns, about 50-90 microns, about 50-100 microns,about 50-250 microns, about 50-500 microns, about 50-750 microns, about50-1,000 microns, about 60-70 microns, about 60-80 microns, about 60-90microns, about 60-100 microns, about 60-250 microns, about 60-500microns, about 60-750 microns, about 60-1,000 microns, about 70-80microns about 70-90 microns, about 70-90 microns, about 70-100 microns,about 70-250 microns, about 70-500 microns, about 70-750 microns, about70-1,000 microns, about 80-90 microns, about 80-100 microns, about80-250 microns, about 80-500 microns, about 80-500 microns, about 80-750microns, about 80-1,000 microns, about 90-100 microns, about 90-250microns, about 90-500 microns, about 90-750 microns, about 90-1,000microns, about 100-250 microns, about 100-500 microns, about 100-750microns, about 100-1,000 microns, about 250-500 microns, about 250-750microns, about 250-1,000 microns, about 500-750 microns, about 500-1,000microns, or about 750-1,000 microns.

In some embodiments, the microbes or compositions comprising themicrobes are combined with a wax, fat, oil, fatty acid, or fattyalcohol, and spray congealed into beads of about 10 microbes, about 20microns, about 30 microns, about 40 microns, about 50 microns, about 60microns, about 70 microns, about 80 microns, about 90 microns, about 100microns, about 150 microns, about 200 microns, about 250 microns, about300 microns, about 350 microns, about 400 microns, about 450 microns,about 500 microns, about 550 microns, about 600 microns, about 650microns, about 700 microns, about 750 microns, about 800 microns, about850 microns, about 900 microns, about 950 microns, or about 1,000microns.

In some embodiments, the microbes or compositions comprising themicrobes are combined with a wax, fat, oil, fatty acid, or fattyalcohol, and spray congealed into beads of between 10-20 microns, 10-30microns, 10-40 microns, 10-50 microns, 10-60 microns, 10-70 microns,10-80 microns, 10-90 microns, 10-100 microns, 10-250 microns, 10-500microns, 10-750 microns, 10-1,000 microns, 20-30 microns, 20-40 microns,20-50 microns, 20-60 microns, 20-70 microns, 20-80 microns, 20-90microns, 20-100 microns, 20-250 microns, 20-500 microns, 20-750 microns,20-1,000 microns, 30-40 microns, 30-50 microns, 30-60 microns, 30-70microns, 30-80 microns, 30-90 microns, 30-100 microns, 30-250 microns,30-500 microns, 30-750 microns, 30-1,000 microns, 40-50 microns, 40-60microns, 40-70 microns, 40-80 microns, 40-90 microns, 40-100 microns,40-250 microns, 40-500 microns, 40-750 microns, 40-1,000 microns, 50-60microns, 50-70 microns, 50-80 microns, 50-90 microns, 50-100 microns,50-250 microns, 50-500 microns, 50-750 microns, 50-1,000 microns, 60-70microns, 60-80 microns, 60-90 microns, 60-100 microns, 60-250 microns,60-500 microns, 60-750 microns, 60-1,000 microns, 70-80 microns 70-90microns, 70-90 microns, 70-100 microns, 70-250 microns, 70-500 microns,70-750 microns, 70-1,000 microns, 80-90 microns, 80-100 microns, 80-250microns, 80-500 microns, 80-500 microns, 80-750 microns, 80-1,000microns, 90-100 microns, 90-250 microns, 90-500 microns, 90-750 microns,90-1,000 microns, 100-250 microns, 100-500 microns, 100-750 microns,100-1,000 microns, 250-500 microns, 250-750 microns, 250-1,000 microns,500-750 microns, 500-1,000 microns, or 750-1,000 microns.

In some embodiments, the microbes or compositions comprising themicrobes are combined with a wax, fat, oil, fatty acid, or fattyalcohol, and spray congealed into beads of between about 10-20 microns,about 10-30 microns, about 10-40 microns, about 10-50 microns, about10-60 microns, about 10-70 microns, about 10-80 microns, about 10-90microns, about 10-100 microns, about 10-250 microns, about 10-500microns, about 10-750 microns, about 10-1,000 microns, about 20-30microns, about 20-40 microns, about 20-50 microns, about 20-60 microns,about 20-70 microns, about 20-80 microns, about 20-90 microns, about20-100 microns, about 20-250 microns, about 20-500 microns, about 20-750microns, about 20-1,000 microns, about 30-40 microns, about 30-50microns, about 30-60 microns, about 30-70 microns, about 30-80 microns,about 30-90 microns, about 30-100 microns, about 30-250 microns, about30-500 microns, about 30-750 microns, about 30-1,000 microns, about40-50 microns, about 40-60 microns, about 40-70 microns, about 40-80microns, about 40-90 microns, about 40-100 microns, about 40-250microns, about 40-500 microns, about 40-750 microns, about 40-1,000microns, about 50-60 microns, about 50-70 microns, about 50-80 microns,about 50-90 microns, about 50-100 microns, about 50-250 microns, about50-500 microns, about 50-750 microns, about 50-1,000 microns, about60-70 microns, about 60-80 microns, about 60-90 microns, about 60-100microns, about 60-250 microns, about 60-500 microns, about 60-750microns, about 60-1,000 microns, about 70-80 microns about 70-90microns, about 70-90 microns, about 70-100 microns, about 70-250microns, about 70-500 microns, about 70-750 microns, about 70-1,000microns, about 80-90 microns, about 80-100 microns, about 80-250microns, about 80-500 microns, about 80-500 microns, about 80-750microns, about 80-1,000 microns, about 90-100 microns, about 90-250microns, about 90-500 microns, about 90-750 microns, about 90-1,000microns, about 100-250 microns, about 100-500 microns, about 100-750microns, about 100-1,000 microns, about 250-500 microns, about 250-750microns, about 250-1,000 microns, about 500-750 microns, about 500-1,000microns, or about 750-1,000 microns.

In some embodiments, the microbes or compositions comprising themicrobes are combined with a wax, fat, oil, fatty acid, or fatty alcoholas well as a water-soluble polymer, salt, polysaccharide, sugar,polypeptide, protein, or sugar alcohol and spray congealed into beads,the size of which are described herein. In some embodiments, thewater-soluble polymer, salt, polysaccharide, sugar, or sugar alcoholserves as a disintegrant. In some embodiments, the disintegrant formspores once the beads are dispersed in the rumen of the animal.

In some embodiments, the composition of the water-soluble polymer, salt,polysaccharide, sugar, polypeptide, protein, or sugar alcohol ismodified such that the disintegrant dissolves within 1, 5, 10, 15, 20,25, 30, 35, 40, 45, 50, 55, 60 minutes of being administered. In someembodiments, the composition of the water-soluble polymer, salt,polysaccharide, sugar, polypeptide, protein, or sugar alcohol ismodified such that the disintegrant dissolves within about 1, about 5,about 10, about 15, about 20, about 25, about 30, about 35, about 40,about 45, about 50, about 55, or about 60 minutes of being administered.

In some embodiments, the composition of the water-soluble polymer, salt,polysaccharide, sugar, polypeptide, protein, or sugar alcohol ismodified such that the disintegrant dissolves within 1, 1.5, 2, 2.5, 3,3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, 9.5, 10, 10.5, 11, 11.5,or 12 hours of being administered. In some embodiments, the compositionof the water-soluble polymer, salt, polysaccharide, sugar, polypeptide,protein, or sugar alcohol is modified such that the disintegrantdissolves within about 1, about 1.5, about 2, about 2.5, about 3, about3.5, about 4, about 4.5, about 5, about 5.5, about 6, about 6.5, about7, about 7.5, about 8, about 8.5, about 9, about 9.5, about 10, about10.5, about 11, about 11.5, or about 12 hours of being administered.

In some embodiments, the composition of the water-soluble polymer, salt,polysaccharide, sugar, polypeptide, protein, or sugar alcohol ismodified such that the disintegrant dissolves at a temperature of atleast 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25,26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43,44, 45, 46, 47, 48, 49, or 50° C. In some embodiments, the compositionof the water-soluble polymer, salt, polysaccharide, sugar, polypeptide,protein, or sugar alcohol is modified such that the disintegrantdissolves at a temperature of at least about 10, least about 11, leastabout 12, least about 13, least about 14, least about 15, least about16, least about 17, least about 18, least about 19, least about 20,least about 21, least about 22, least about 23, least about 24, leastabout 25, least about 26, least about 27, least about 28, least about29, least about 30, least about 31, least about 32, least about 33,least about 34, about 35, about 36, about 37, about 38, about 39, about40, about 41, about 42, about 43, about 44, least about 45, least about46, least about 47, least about 48, least about 49, or least about 50°C.

In some embodiments, the composition of the water-soluble polymer, salt,polysaccharide, sugar, polypeptide, protein, or sugar alcohol ismodified such that the disintegrant dissolves at a pH of at least 3.8,3.9, 4. 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, 5.0, 5.1, 5.2, 5.3,5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6.0, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8,6.9, 7.0, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9, 8.0, 8.1, 8.2,8.3, 8.4, 8.5, 8.6, 8.7, 8.8, 8.9, 9.0, 9.1, 9.2, 9.3, 9.4, 9.5, 9.6,9.7, 9.8, 9.9 or 10.0. In some embodiments, the composition of thewater-soluble polymer, salt, polysaccharide, sugar, polypeptide,protein, or sugar alcohol is modified such that the disintegrantdissolves at a pH of at least about 3.8, least about 3.9, least about 4.least about 4.1, least about 4.2, least about 4.3, least about 4.4,least about 4.5, least about 4.6, least about 4.7, least about 4.8,least about 4.9, least about 5.0, least about 5.1, least about 5.2,least about 5.3, least about 5.4, least about 5.5, least about 5.6,least about 5.7, least about 5.8, least about 5.9, least about 6.0,least about 6.2, least about 6.3, least about 6.4, least about 6.5,least about 6.6, least about 6.7, least about 6.8, least about 6.9,least about 7.0, least about 7.1, least about 7.2, least about 7.3,least about 7.4, least about 7.5, least about 7.6, least about 7.7,least about 7.8, least about 7.9, least about 8.0, least about 8.1,least about 8.2, least about 8.3, least about 8.4, least about 8.5,least about 8.6, least about 8.7, least about 8.8, least about 8.9,least about 9.0, least about 9.1, least about 9.2, least about 9.3,least about 9.4, least about 9.5, least about 9.6, least about 9.7,least about 9.8, least about 9.9, or least about 10.0.

In some embodiments, the microbes or compositions comprising themicrobes are coated with a polymer, a polysaccharide, sugar, sugaralcohol, gel, wax, fat, fatty alcohol, or fatty acid.

In some embodiments, the microbes or compositions comprising themicrobes are coated with a polymer, a polysaccharide, sugar, sugaralcohol, gel, wax, fat, fatty alcohol, or fatty acid.

In some embodiments, the coating of the microbes or compositionscomprising the microbes is modified such that the coating dissolveswithin 1, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60 minutes of beingadministered. In some embodiments, the coating of the microbes orcompositions comprising the microbes is modified such that the coatingdissolves within about 1, about 5, about 10, about 15, about 20, about25, about 30, about 35, about 40, about 45, about 50, about 55, or about60 minutes of being administered.

In some embodiments, the coating of the microbes or compositionscomprising the microbes is modified such that the coating dissolveswithin 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5,9, 9.5, 10, 10.5, 11, 11.5, or 12 hours of being administered. In someembodiments, the coating of the microbes or compositions comprising themicrobes is modified such that the coating dissolves within about 1,about 1.5, about 2, about 2.5, about 3, about 3.5, about 4, about 4.5,about 5, about 5.5, about 6, about 6.5, about 7, about 7.5, about 8,about 8.5, about 9, about 9.5, about 10, about 10.5, about 11, about11.5, or about 12 hours of being administered.

In some embodiments, the coating of the microbes or compositionscomprising the microbes is modified such that the coating dissolves at atemperature of at least 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21,22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39,40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50° C. In some embodiments,the coating of the microbes or compositions comprising the microbes ismodified such that the coating dissolves at a temperature of at leastabout 10, least about 11, least about 12, least about 13, least about14, least about 15, least about 16, least about 17, least about 18,least about 19, least about 20, least about 21, least about 22, leastabout 23, least about 24, least about 25, least about 26, least about27, least about 28, least about 29, least about 30, least about 31,least about 32, least about 33, least about 34, about 35, about 36,about 37, about 38, about 39, about 40, about 41, about 42, about 43,about 44, least about 45, least about 46, least about 47, least about48, least about 49, or least about 50° C.

In some embodiments, the coating of the microbes or compositionscomprising the microbes is modified such that the coating dissolves at apH of at least 3.8, 3.9, 4. 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9,5.0, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6.0, 6.2, 6.3, 6.4,6.5, 6.6, 6.7, 6.8, 6.9, 7.0, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8,7.9, 8.0, 8.1, 8.2, 8.3, 8.4, 8.5, 8.6, 8.7, 8.8, 8.9, 9.0, 9.1, 9.2,9.3, 9.4, 9.5, 9.6, 9.7, 9.8, 9.9 or 10.0. In some embodiments, thecoating of the microbes or compositions comprising the microbes ismodified such that the coating dissolves at a pH of at least about 3.8,least about 3.9, least about 4. least about 4.1, least about 4.2, leastabout 4.3, least about 4.4, least about 4.5, least about 4.6, leastabout 4.7, least about 4.8, least about 4.9, least about 5.0, leastabout 5.1, least about 5.2, least about 5.3, least about 5.4, leastabout 5.5, least about 5.6, least about 5.7, least about 5.8, leastabout 5.9, least about 6.0, least about 6.2, least about 6.3, leastabout 6.4, least about 6.5, least about 6.6, least about 6.7, leastabout 6.8, least about 6.9, least about 7.0, least about 7.1, leastabout 7.2, least about 7.3, least about 7.4, least about 7.5, leastabout 7.6, least about 7.7, least about 7.8, least about 7.9, leastabout 8.0, least about 8.1, least about 8.2, least about 8.3, leastabout 8.4, least about 8.5, least about 8.6, least about 8.7, leastabout 8.8, least about 8.9, least about 9.0, least about 9.1, leastabout 9.2, least about 9.3, least about 9.4, least about 9.5, leastabout 9.6, least about 9.7, least about 9.8, least about 9.9, or leastabout 10.0.

Animal Feed

In some embodiments, compositions of the present disclosure are mixedwith animal feed. In some embodiments, animal feed may be present invarious forms such as pellets, capsules, granulated, powdered, mash,liquid, semi-liquid, or mixed rations(s).

In some embodiments, compositions of the present disclosure are mixedinto the premix at the feed mill (e.g., Carghill or Western Millin),alone as a standalone premix, and/or alongside other feed additives suchas MONENSIN, vitamins, etc. In one embodiment, compositions of thepresent disclosure are mixed into the feed itself. In one embodiment,the compositions of the present disclosure are mixed into the feed atthe feed mill.

In some embodiments, feed of the present disclosure may be supplementedwith water, premix or premixes, forage, beans (e.g., whole, cracked, orground), grains (e.g., whole, cracked, or ground), bean- or grain-basedoils, bean- or grain-based meals, bean- or grain-based haylage orsilage, bean- or grain-based syrups, fatty acids, sugar alcohols (e.g.,polyhydric alcohols), commercially available formula feeds, oystershells and those of other bivalves, and mixtures thereof.

In some embodiments, forage encompasses hay, haylage, and silage. Insome embodiments, hays include grass hays (e.g., sudangrass,orchardgrass, or the like), alfalfa hay, and clover hay. In someembodiments, haylages include grass haylages, sorghum haylage, andalfalfa haylage. In some embodiments, silages include maize, oat, wheat,alfalfa, clover, and the like.

In some embodiments, premix or premixes may be utilized in the feed.Premixes may comprise micro-ingredients such as vitamins, minerals,amino acids; chemical preservatives; pharmaceutical compositions such asantibiotics, ionophores, and other medicaments; fermentation products,and other ingredients. In some embodiments, premixes are blended intothe feed.

In some embodiments, the feed may include feed concentrates such assoybean hulls, soybean oils, sugar beet pulp, molasses, high proteinsoybean meal, ground corn, shelled corn, cornflakes, wheat midds,distiller grain, cottonseed hulls, rumen-bypass protein, rumen-bypassfat, and grease. See Luhman (U.S. Publication US20150216817A1), Andersonet al. (U.S. Pat. No. 3,484,243), Porter and Luhman (U.S. Pat. No.9,179,694B2), Iritani et al. (U.S. Pat. No. 6,090,416), Axelrod et al.(U.S. Publication US20060127530A1), and Katsumi et al. (U.S. Pat. No.5,741,508) for animal feed and animal feed supplements capable of use inthe present compositions and methods.

In some embodiments, feed occurs as a compound, which includes, in amixed composition capable of meeting the basic dietary needs, the feeditself, vitamins, minerals, amino acids, and other necessary components.Compound feed may further comprise premixes.

In some embodiments, microbial compositions of the present disclosuremay be mixed with animal feed, premix, and/or compound feed. Individualcomponents of the animal feed may be mixed with the microbialcompositions prior to feeding to beef cattle. The microbial compositionsof the present disclosure may be applied into or on a premix, into or ona feed, and/or into or on a compound feed.

In some embodiments, microbial compositions of the present disclosuremay be mixed with animal feed, premix, and/or compound feed at variousstages of animal adaptation to the step-up or finishing diet.

In some embodiments, microbial compositions of the present disclosureare mixed with feed and microingredients. Microingredients includeliquid fat blends, glycerin, rumensin, monensin, vitamins, tylan,optaflex, melengesterol acetate, minerals, and amino acids. In someembodiments, the mixing of feed, microbial compositions of the presentdisclosure, and microingredients is performed at the feedlot.

In some embodiments, cattle begin a step up or a starting ration. Asused herein, a “step-up diet” or “starting ration” is a diet fed tofeedlot cattle as a transition to the high grain content of thefinishing diet. In some embodiments, the step-up diet may involve one ormore step-up diets that ease the cattle into the transition to thefinishing diet. In some embodiments, the step-up diet is formulated toslowly increase the amount of high energy feed in the diet whilemitigating gastrointestinal distress and the effects of rapid onsetacidosis. In some embodiments, the cattle are fed a single type ofstep-up diet. In some embodiments, the cattle are fed multiple varietiesof step-up diets, increasing the amount of high energy feed with eachiteration of the step up diet variety. In some embodiments, the cattleare fed at least one step up diet, wherein the subsequent diets aredifferent from each of those step up diets that follow. In someembodiments, the cattle are fed at least two different step up diets. Insome embodiments, the cattle are fed at least three different step updiets.

As used herein, a “finishing diet” is a concentrated high-energy diet(often high-grain) fed to cattle on a feedlot to rapidly bring thecattle up to get them to market weight by the time the cattle arerendered. In some embodiments, the finishing diet may result in liverdisease, liver abscesses, and/or acidosis.

In some embodiments, the microbial compositions of the presentdisclosure are mixed with step-up diets. In some embodiments, themicrobial compositions of the present disclosure are mixed withfinishing diets.

Administration of Microbial Compositions

In some embodiments, the microbial compositions of the presentdisclosure are administered to cattle via the oral route. In someembodiments the microbial compositions are administered via a directinjection route into the gastrointestinal tract. In further embodiments,the direct injection administration delivers the microbial compositionsdirectly to the rumen. In some embodiments, the microbial compositionsof the present disclosure are administered to animals anally. In furtherembodiments, anal administration is in the form of an insertedsuppository.

In some embodiments, the microbial composition is administered in a dosevolume comprising a total of, or at least, 1 ml, 2 ml, 3 ml, 4 ml, 5 ml,6 ml, 7 ml, 8 ml, 9 ml, 10 ml, llml, 12 ml, 13 ml, 14 ml, 15 ml, 16 ml,17 ml, 18 ml, 19 ml, 20 ml, 21 ml, 22 ml, 23 ml, 24 ml, 25 ml, 26 ml, 27ml, 28 ml, 29 ml, 30 ml, 31 ml, 32 ml, 33 ml, 34 ml, 35 ml, 36 ml, 37ml, 38 ml, 39 ml, 40 ml, 41m, 42 ml, 43 ml, 44 ml, 45 ml, 46 ml, 47 ml,48 ml, 49 ml, 50 ml, 60 ml, 70 ml, 80 ml, 90 ml, 100 ml, 200 ml, 300 ml,400 ml, 500 ml, 600 ml, 700 ml, 800 ml, 900 ml, or 1,000 ml.

In some embodiments, the microbial composition is administered in a dosecomprising a total of, or at least, 10¹⁸, 10¹⁷, 10¹⁶, 10¹⁵, 10¹⁴, 10¹³,10¹², 10¹¹, 10¹⁰, 10⁹, 10⁸, 10⁷, 10⁶, 10⁵, 10⁴, 10³, or 10² microbialcells.

In some embodiments, the microbial compositions are mixed with feed, andthe administration occurs through the ingestion of the microbialcompositions along with the feed. In some embodiments, the dose of themicrobial composition is administered such that there exists 10² to10¹², 10³ to 10¹², 10⁴ to 10¹², 10⁵ to 10¹², 10⁶ to 10¹², 10⁷ to 10¹²,10⁸ to 10¹², 10⁹ to 10¹², 10¹⁰ to 10¹², 10¹¹ to 10¹², 10² to 10¹¹, 10³to 10¹¹, 10⁴ to 10¹¹, 10⁵ to 10¹¹, 10⁶ to 10¹¹, 10⁷ to 10¹¹, 10⁸ to10¹¹, 10⁹ to 10¹¹, 10¹⁰ to 10¹¹, 10² to 10¹⁰, 10³ to 10¹⁰, 10⁴ to 10¹⁰,10⁵ to 10¹⁰, 10⁶ to 10¹⁰, 10⁷ to 10¹⁰, 10⁸ to 10¹⁰, 10⁹ to 10¹⁰, 10² to10⁹, 10³ to 10⁹, 10⁴ to 10⁹, 10⁵ to 10⁹, 10⁶ to 10⁹, 10⁷ to 10⁹, 10⁸ to10⁹, 10² to 10⁸, 10³ to 10⁸, 10⁴ to 10⁸, 10⁵ to 10⁸, 10⁶ to 10⁸, 10⁷ to10⁸, 10² to 10⁷, 10³ to 10⁷, 10⁴ to 10⁷, 10⁵ to 10⁷, 10⁶ to 10⁷, 10² to10⁶, 10³ to 10⁶, 10⁴ to 10⁶, 10⁵ to 10⁶, 10² to 10⁵, 10³ to 10⁵, 10⁴ to10⁵, 10² to 10⁴, 10³ to 10⁴, 10² to 10³, 10¹², 10¹¹, 10¹⁰, 10⁹, 10⁸,10⁷, 10⁶, 10⁵, 10⁴, 10³, or10² total microbial cells per gram ormilliliter of the composition.

In some embodiments, the administered dose of the microbial compositioncomprises 10² to 10¹⁸, 10³ to 10¹⁸, 10⁴ to 10¹⁸, 10⁵ to 10¹⁸, 10⁶ to10¹⁸, 10⁷ to 10¹⁸, 10⁸ to 10¹⁸, 10⁹ to 10¹⁸, 10¹⁰ to 10¹⁸, 10¹¹ to 10¹⁸,10¹² to 10¹⁸, 10¹³ to 10¹⁸, 10¹⁴ to 10¹⁸, 10¹⁵ to 10¹⁸, 10¹⁶ to 10¹⁸,10¹⁷ to 10¹⁸, 10² to 10¹², 10³ to 10¹², 10⁴ to 10¹², 10⁵ to 10¹², 10⁶ to10¹², 10⁷ to 10¹², 10⁸ to 10¹², 10⁹ to 10¹², 10¹⁰ to 10¹², 10¹¹ to 10¹²,10² to 10¹¹, 10³ to 10¹¹, 10⁴ to 10¹¹, 10⁵ to 10¹¹, 10 ⁶ to 10¹¹, 10⁷ to10¹¹, 10 ⁸ to 10¹¹, 10⁹ to 10¹¹, 10¹⁰ to 10¹¹, 10² to 10¹⁰, 10³ to 10¹⁰,10⁴ to 10¹⁰, 10⁵ to 10¹⁰, 10⁶ to 10¹⁰, 10⁷ to 10¹⁰, 10⁸ to 10¹⁰, 10⁹ to10¹⁰, 10² to 10⁹, 10³ to 10⁹, 10⁴ to 10⁹, 10⁵ to 10⁹, 10⁶ to 10⁹, 10⁷ to10⁹, 10⁸ to 10⁹, 10² to 10⁸, 10³ to 10⁸, 10⁴ to 10⁸, 10⁵ to 10⁸, 10⁶ to10⁸, 10⁷ to 10⁸, 10² to 10⁷, 10³ to 10⁷, 10⁴ to 10⁷, 10⁵ to 10⁷, 10⁶ to10⁷, 10² to 10⁶, 10³ to 10⁶, 10⁴ to 10⁶, 10⁵ to 10⁶ 10² to 10⁵, 10³ to10⁵, 10⁴ to 10⁵, 10² to 10⁴, 10³ to 10⁴, 10² to 10³, 10¹⁸, 10¹⁷, 10¹⁶,10¹⁵, 10¹⁴, 10¹³, 10¹², 10¹¹, 10 ¹⁰, 10⁹, 10⁸, 10⁷, 10⁶, 10⁵, 10⁴, 10³,or 10² total microbial cells.

In some embodiments, the composition is administered 1 or more times perday. In some aspects, the composition is administered with food eachtime the animal is fed. In some embodiments, the composition isadministered 1 to 10, 1 to 9, 1 to 8, 1 to 7, 1 to 6, 1 to 5, 1 to 4, 1to 3, 1 to 2, 2 to 10, 2 to 9, 2 to 8, 2 to 7, 2 to 6, 2 to 5, 2 to 4, 2to 3, 3 to 10, 3 to 9, 3 to 8, 3 to 7, 3 to 6, 3 to 5, 3 to 4, 4 to 10,4 to 9, 4 to 8, 4 to 7, 4 to 6, 4 to 5, 5 to 10, 5 to 9, 5 to 8, 5 to 7,5 to 6, 6 to 10, 6 to 9, 6 to 8, 6 to 7, 7 to 10, 7 to 9, 7 to 8, 8 to10, 8 to 9, 9 to 10, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 times per day.

In some embodiments, the microbial composition is administered 1 to 10,1 to 9, 1 to 8, 1 to 7, 1 to 6, 1 to 5, 1 to 4, 1 to 3, 1 to 2, 2 to 10,2 to 9, 2 to 8, 2 to 7, 2 to 6, 2 to 5, 2 to 4, 2 to 3, 3 to 10, 3 to 9,3 to 8, 3 to 7, 3 to 6, 3 to 5, 3 to 4, 4 to 10, 4 to 9, 4 to 8, 4 to 7,4 to 6, 4 to 5, 5 to 10, 5 to 9, 5 to 8, 5 to 7, 5 to 6, 6 to 10, 6 to9, 6 to 8, 6 to 7, 7 to 10, 7 to 9, 7 to 8, 8 to 10, 8 to 9, 9 to 10, 1,2, 3, 4, 5, 6, 7, 8, 9, or 10 times per week.

In some embodiments, the microbial composition is administered 1 to 10,1 to 9, 1 to 8, 1 to 7, 1 to 6, 1 to 5, 1 to 4, 1 to 3, 1 to 2, 2 to 10,2 to 9, 2 to 8, 2 to 7, 2 to 6, 2 to 5, 2 to 4, 2 to 3, 3 to 10, 3 to 9,3 to 8, 3 to 7, 3 to 6, 3 to 5, 3 to 4, 4 to 10, 4 to 9, 4 to 8, 4 to 7,4 to 6, 4 to 5, 5 to 10, 5 to 9, 5 to 8, 5 to 7, 5 to 6, 6 to 10, 6 to9, 6 to 8, 6 to 7, 7 to 10, 7 to 9, 7 to 8, 8 to 10, 8 to 9, 9 to 10, 1,2, 3, 4, 5, 6, 7, 8, 9, or 10 times per month.

In some embodiments, the microbial composition is administered 1 to 10,1 to 9, 1 to 8, 1 to 7, 1 to 6, 1 to 5, 1 to 4, 1 to 3, 1 to 2, 2 to 10,2 to 9, 2 to 8, 2 to 7, 2 to 6, 2 to 5, 2 to 4, 2 to 3, 3 to 10, 3 to 9,3 to 8, 3 to 7, 3 to 6, 3 to 5, 3 to 4, 4 to 10, 4 to 9, 4 to 8, 4 to 7,4 to 6, 4 to 5, 5 to 10, 5 to 9, 5 to 8, 5 to 7, 5 to 6, 6 to 10, 6 to9, 6 to 8, 6 to 7, 7 to 10, 7 to 9, 7 to 8, 8 to 10, 8 to 9, 9 to 10, 1,2, 3, 4, 5, 6, 7, 8, 9, or 10 times per year.

In some embodiments, the microbial composition is administered toanimals throughout the entire time they are on the feedlot. In someembodiments, the microbial composition is administered to animals onlyduring a portion of time while they are on the feedlot. In someembodiments, the microbial composition is administered only during thegrower phase. In some embodiments, the microbial composition isadministered only during the time when animals are in the receiving pen.In some embodiments, the microbial composition is administered only whenthe animals are receiving vaccinations and/or treatments. In someembodiments, the microbial composition is administered only when theanimals are on a step up diet or when being adapted to a high graindiet. In some embodiments, the microbial composition is administeredonly when the animals are on a finisher diet or a high grain diet.

In some embodiments, the microbial composition is administered duringthe grower phase, when animals are in the receiving pen, when animalsare receiving vaccinations and/or treatments, when animals are beingadapted to a high grain diet or are on a step up diet, and/or when theanimals are on a finisher diet or a high grain diet.

In some embodiments, an animal entering the feed lot receives at leastone microbial composition prior to entering the feed lot. In someembodiments, an animal on the feed lot receives a microbial compositionthat is different from the first at least one microbial composition. Infurther embodiments, an animal on the feed lot receives a microbialcomposition that is different from the first and second at least onemicrobial composition.

In some embodiments, the type of diet fed to the animal corresponds withthe type of microbial composition administered to the animal. In someembodiments, a grazing or grass/hay-fed animal will receive a firstmicrobial composition. In further embodiments, the same animal fed adifferent diet will receive a second microbial composition, wherein thefirst microbial composition is different from the second microbialcomposition. In some embodiments, the same animal fed yet a differentdiet will receive a third microbial composition, wherein the firstmicrobial composition is different from the second and third microbialcompositions. In some embodiments, the same animal fed yet a differentdiet will receive a fourth microbial composition, wherein the firstmicrobial composition is different from the second, third, and fourthmicrobial compositions. In some embodiments, the same animal fed yet adifferent diet will receive a fifth microbial composition, wherein thefirst microbial composition is different from the second, third, fourth,and fifth microbial compositions.

In some embodiments, the feed can be uniformly coated with one or morelayers of the microbes and/or microbial compositions disclosed herein,using conventional methods of mixing, spraying, or a combination thereofthrough the use of treatment application equipment that is specificallydesigned and manufactured to accurately, safely, and efficiently applycoatings. Such equipment uses various types of coating technology suchas rotary coaters, drum coaters, fluidized bed techniques, spouted beds,rotary mists, or a combination thereof. Liquid treatments such as thoseof the present disclosure can be applied via either a spinning“atomizer” disk or a spray nozzle, which evenly distributes themicrobial composition onto the feed as it moves though the spraypattern. In some aspects, the feed is then mixed or tumbled for anadditional period of time to achieve additional treatment distributionand drying.

In some embodiments, the feed coats of the present disclosure can be upto 10 μm, 20 μm, 30 μm, 40 μm, 50 μm, 60 μm, 70 μm, 80 μm, 90 μm, 100μm, 110 μm, 120 μm, 130 μm, 140 μm, 150 μm, 160 μm, 170 μm, 180 μm, 190μm, 200 μm, 210 μm, 220 μm, 230 μm, 240 μm, 250 μm, 260 μm, 270 μm, 280μm, 290 μm, 300 μm, 310 μm, 320 μm, 330 μm, 340 μm, 350 μm, 360 μm, 370μm, 380 μm, 390 μm, 400 μm, 410 μm, 420 μm, 430 μm, 440 μm, 450 μm, 460μm, 470 μm, 480 μm, 490 μm, 500 μm, 510 μm, 520 μm, 530 μm, 540 μm, 550μm, 560 μm, 570 μm, 580 μm, 590 μm, 600 μm, 610 μm, 620 μm, 630 μm, 640μm, 650 μm, 660 μm, 670 μm, 680 μm, 690 μm, 700 μm, 710 μm, 720 μm, 730μm, 740 μm, 750 μm, 760 μm, 770 μm, 780 μm, 790 μm, 800 μm, 810 μm, 820μm, 830 μm, 840 μm, 850 μm, 860 μm, 870 μm, 880 μm, 890 μm, 900 μm, 910μm, 920 μm, 930 μm, 940 μm, 950 μm, 960 μm, 970 μm, 980 μm, 990 μm, 1000μm, 1010 μm, 1020 μm, 1030 μm, 1040 μm, 1050 μm, 1060 μm, 1070 μm, 1080μm, 1090 μm, 1100 μm, 1110 μm, 1120 μm, 1130 μm, 1140 μm, 1150 μm, 1160μm, 1170 μm, 1180 μm, 1190 μm, 1200 μm, 1210 μm, 1220 μm, 1230 μm, 1240μm, 1250 μm, 1260 μm, 1270 μm, 1280 μm, 1290 μm, 1300 μm, 1310 μm, 1320μm, 1330 μm, 1340 μm, 1350 μm, 1360 μm, 1370 μm, 1380 μm, 1390 μm, 1400μm, 1410 μm, 1420 μm, 1430 μm, 1440 μm, 1450 μm, 1460 μm, 1470 μm, 1480μm, 1490 μm, 1500 μm, 1510 μm, 1520 μm, 1530 μm, 1540 μm, 1550 μm, 1560μm, 1570 μm, 1580 μm, 1590 μm, 1600 μm, 1610 μm, 1620 μm, 1630 μm, 1640μm, 1650 μm, 1660 μm, 1670 μm, 1680 μm, 1690 μm, 1700 μm, 1710 μm, 1720μm, 1730 μm, 1740 μm, 1750 μm, 1760 μm, 1770 μm, 1780 μm, 1790 μm, 1800μm, 1810 μm, 1820 μm, 1830 μm, 1840 μm, 1850 μm, 1860 μm, 1870 μm, 1880μm, 1890 μm, 1900 μm, 1910 μm, 1920 μm, 1930 μm, 1940 μm, 1950 μm, 1960μm, 1970 μm, 1980 μm, 1990 μm, 2000 μm, 2010 μm, 2020 μm, 2030 μm, 2040μm, 2050 μm, 2060 μm, 2070 μm, 2080 μm, 2090 μm, 2100 μm, 2110 μm, 2120μm, 2130 μm, 2140 μm, 2150 μm, 2160 μm, 2170 μm, 2180 μm, 2190 μm, 2200μm, 2210 μm, 2220 μm, 2230 μm, 2240 μm, 2250 μm, 2260 μm, 2270 μm, 2280μm, 2290 μm, 2300 μm, 2310 μm, 2320 μm, 2330 μm, 2340 μm, 2350 μm, 2360μm, 2370 μm, 2380 μm, 2390 μm, 2400 μm, 2410 μm, 2420 μm, 2430 μm, 2440μm, 2450 μm, 2460 μm, 2470 μm, 2480 μm, 2490 μm, 2500 μm, 2510 μm, 2520μm, 2530 μm, 2540 μm, 2550 μm, 2560 μm, 2570 μm, 2580 μm, 2590 μm, 2600μm, 2610 μm, 2620 μm, 2630 μm, 2640 μm, 2650 μm, 2660 μm, 2670 μm, 2680μm, 2690 μm, 2700 μm, 2710 μm, 2720 μm, 2730 μm, 2740 μm, 2750 μm, 2760μm, 2770 μm, 2780 μm, 2790 μm, 2800 μm, 2810 μm, 2820 μm, 2830 μm, 2840μm, 2850 μm, 2860 μm, 2870 μm, 2880 μm, 2890 μm, 2900 μm, 2910 μm, 2920μm, 2930 μm, 2940 μm, 2950 μm, 2960 μm, 2970 μm, 2980 μm, 2990 μm, or3000 μm thick.

In some embodiments, the microbial cells can be coated freely onto anynumber of compositions or they can be formulated in a liquid or solidcomposition before being coated onto a composition. For example, a solidcomposition comprising the microorganisms can be prepared by mixing asolid carrier with a suspension of the spores until the solid carriersare impregnated with the spore or cell suspension. This mixture can thenbe dried to obtain the desired particles.

In some other embodiments, it is contemplated that the solid or liquidmicrobial compositions of the present disclosure further containfunctional agents e.g., activated carbon, minerals, vitamins, and otheragents capable of improving the quality of the products or a combinationthereof.

Methods of coating and compositions in use of said methods that areknown in the art can be particularly useful when they are modified bythe addition of one of the embodiments of the present disclosure. Suchcoating methods and apparatus for their application are disclosed in,for example: U.S. Pat. Nos. 8,097,245 and 7,998,502; and PCT Pat. App.Pub. Nos. WO 2008/076975, WO 2010/138522, WO 2011/094469, WO2010/111347, and WO 2010/111565 each of which is incorporated byreference herein.

In some embodiments, the microbes or microbial compositions of thepresent disclosure exhibit a synergistic effect, on one or more of thetraits described herein, in the presence of one or more of the microbesor microbial compositions coming into contact with one another. Thesynergistic effect obtained by the taught methods can be quantified, forexample, according to Colby's formula (i.e., (E)=X+Y−(X*Y/100)). SeeColby, R. S., “Calculating Synergistic and Antagonistic Responses ofHerbicide Combinations,” 1967. Weeds. Vol. 15, pp. 20-22, incorporatedherein by reference in its entirety. Thus, “synergistic” is intended toreflect an outcome/parameter/effect that has been increased by more thanan additive amount.

In some embodiments, the microbes or microbial compositions of thepresent disclosure may be administered via bolus. In one embodiment, abolus (e.g., capsule containing the composition) is inserted into abolus gun, and the bolus gun is inserted into the buccal cavity and/oresophagus of the animal, followed by the release/injection of the bolusinto the animal's digestive tract. In one embodiment, the bolusgun/applicator is a BOVIKALC bolus gun/applicator. In anotherembodiment, the bolus gun/applicator is a QUADRICAL gun/applicator.

In some embodiments, the microbes or microbial compositions of thepresent disclosure may be administered via drench. In one embodiment,the drench is an oral drench. A drench administration comprisesutilizing a drench kit/applicator/syringe that injects/releases a liquidcomprising the microbes or microbial compositions into the buccal cavityand/or esophagus of the animal.

In some embodiments, the microbes or microbial compositions of thepresent disclosure may be administered in a time-released fashion. Thecomposition may be coated in a chemical composition, or may be containedin a mechanical device or capsule that releases the microbes ormicrobial compositions over a period of time instead all at once. In oneembodiment, the microbes or microbial compositions are administered toan animal in a time-release capsule. In one embodiment, the compositionmay be coated in a chemical composition, or may be contained in amechanical device or capsule that releases the microbes or microbialcompositions all at once a period of time hours post ingestion.

In some embodiments, one microbe composition is administered one or moretimes when the animals are on a step up diet, and a different microbecomposition is administered one or more times when the animals are on afinishing diet. In some embodiments, one microbe composition isadministered one or more times when the animals are on a step up diet, adifferent microbe composition is administered one or more times when theanimals are on the first thirty days of the finishing diet, and yet adifferent microbe composition is administered one or more times when theanimals have been on the finishing diet for greater than thirty days.

In some embodiments, one microbe composition is administered one or moretimes while the animals exhibit signs of acidosis, and different microbecomposition is administered one or more times once the signs of acidosishave abated. In some embodiments, a microbe composition is administeredto animals that do not exhibit signs of acidosis, and a differentmicrobe composition is administered if the animals exhibit signs ofacidosis.

In some embodiments, the microbes or microbial compositions areadministered in a time-released fashion between 1 to 5, 1 to 10, 1 to15, 1 to 20, 1 to 24, 1 to 25, 1 to 30, 1 to 35, 1 to 40, 1 to 45, 1 to50, 1 to 55, 1 to 60, 1 to 65, 1 to 70, 1 to 75, 1 to 80, 1 to 85, 1 to90, 1 to 95, or 1 to 100 hours.

In some embodiments, the microbes or microbial compositions areadministered in a time-released fashion between 1 to 2, 1 to 3, 1 to 4,1 to 5, 1 to 6, 1 to 7, 1 to 8, 1 to 9, 1 to 10, 1 to 11, 1 to 12, 1 to13, 1 to 14, 1 to 15, 1 to 16, 1 to 17, 1 to 18, 1 to 19, 1 to 20, 1 to21, 1 to 22, 1 to 23, 1 to 24, 1 to 25, 1 to 26, 1 to 27, 1 to 28, 1 to29, or 1 to 30 days.

Microorganisms

As used herein the term “microorganism” should be taken broadly. Itincludes, but is not limited to, the two prokaryotic domains, Bacteriaand Archaea, as well as eukaryotic fungi, protozoa, and viruses.

By way of example, the microorganisms may include species of the generaof: Fibrobacter, Saccharofermentans, Bacillus, Spirochaeta, Bacteroides,Lachnospiracea incertae sedis, Clostridium XLVa, Ruminococcus,Butyricimonas, Olsenella, Acidaminococcus, Parabacteroides, Clostridumsensu stricto, Oribacterium, Pseudoflavonifractor, Treponema,Rhodobacter, Fluviicola, Succiniclasticum, Solobacterium, Veillonella,Cellulosimicrobium, Cupriavidus, Megasphaera, Succinivibrio,Oscillibacter, Pseudomonas, Corynebacterium, Adlercreutzia, Dorea,Roseburia, Anaerovibrio, Sporosarcina, Streptomyces, Syntrophococcus,Butyrivibrio, Lachnobacterium, Pyramidobacter, Coprococcus,Ruminobacter, Thermobifidia, Papillibacter, Aquimarina, Propioniciclava,Staphylococcus, Mogibacterium, Pseudobutyrivibrio, Asteroleplasma,Turicibacter, Aggregatibacter, Brevundimonas, Phascolarctobacterium, andSphingobium.

In certain embodiments, the microorganism is unculturable. This shouldbe taken to mean that the microorganism is not known to be culturable oris difficult to culture using methods known to one skilled in the art.

In one embodiment, the microbes are obtained from animals (e.g.,mammals, reptiles, birds, and the like), soil (e.g., rhizosphere), air,water (e.g., marine, freshwater, wastewater sludge), sediment, oil,plants (e.g., roots, leaves, stems), agricultural products, and extremeenvironments (e.g., acid mine drainage or hydrothermal systems). In afurther embodiment, microbes obtained from marine or freshwaterenvironments such as an ocean, river, or lake. In a further embodiment,the microbes can be from the surface of the body of water, or any depthof the body of water (e.g., a deep sea sample).

The microorganisms of the disclosure may be isolated in substantiallypure or mixed cultures. They may be concentrated, diluted, or providedin the natural concentrations in which they are found in the sourcematerial. For example, microorganisms from saline sediments may beisolated for use in this disclosure by suspending the sediment in freshwater and allowing the sediment to fall to the bottom. The watercontaining the bulk of the microorganisms may be removed by decantationafter a suitable period of settling and either administered to the GItract of beef cattle, or concentrated by filtering or centrifugation,diluted to an appropriate concentration and administered to the GI tractof beef cattle with the bulk of the salt removed. By way of furtherexample, microorganisms from mineralized or toxic sources may besimilarly treated to recover the microbes for application to beef cattleto minimize the potential for damage to the animal.

In another embodiment, the microorganisms are used in a crude form, inwhich they are not isolated from the source material in which theynaturally reside. For example, the microorganisms are provided incombination with the source material in which they reside; for example,fecal matter, rumen content, rumen fluid, or other composition found inthe gastrointestinal tract. In this embodiment, the source material mayinclude one or more species of microorganisms.

In some embodiments, a mixed population of microorganisms is used in themethods of the disclosure.

In embodiments of the disclosure where the microorganisms are isolatedfrom a source material (for example, the material in which theynaturally reside), any one or a combination of a number of standardtechniques which will be readily known to skilled persons may be used.However, by way of example, these in general employ processes by which asolid or liquid culture of a single microorganism can be obtained in asubstantially pure form, usually by physical separation on the surfaceof a solid microbial growth medium or by volumetric dilutive isolationinto a liquid microbial growth medium. These processes may includeisolation from dry material, liquid suspension, slurries or homogenatesin which the material is spread in a thin layer over an appropriatesolid gel growth medium, or serial dilutions of the material made into asterile medium and inoculated into liquid or solid culture media.

While not essential, in one embodiment, the material containing themicroorganisms may be pre-treated prior to the isolation process inorder to either multiply all microorganisms in the material, removecertain microorganisms in the material, and/or shift the distribution ofmicroorganisms in the material. Microorganisms can then be isolated fromthe enriched materials as disclosed above.

In certain embodiments, as mentioned herein before, the microorganism(s)may be used in crude form and need not be isolated from an animal or amedia. For example, feces, or growth media which includes themicroorganisms identified to be of benefit to increased feed efficiencymay be obtained and used as a crude source of microorganisms for thenext round of the method or as a crude source of microorganisms at theconclusion of the method. For example, fresh feces could be obtained andoptionally processed.

Microbiome Shift and Abundance of Microbes

In some embodiments, the microbiome of beef cattle, including the rumenmicrobiome comprises a diverse arrive of microbes with a wide variety ofmetabolic capabilities. The microbiome is influenced by a range offactors including diet, variations in animal metabolism, and breed,among others. Most cattle diets are plant-based and rich in complexpolysaccharides that enrich the gastrointestinal microbial community formicrobes capable of breaking down specific polymeric components in thediet such as cellulose, hemicellulose, lignin, etc. The end products ofprimary degradation sustain a chain of microbes that ultimately producea range of organic acids together with hydrogen and carbon dioxide.Because of the complex and interlinked nature of the microbiome,changing the diet and thus substrates for primary degradation may have acascading effect on gut microbial metabolism, with changes in both theorganic acid profiles and the methane levels produced, thus impactingthe quality and quantity of animal production and or the productsproduced by the animal. See Menezes et al. (2011. FEMS Microbiol. Ecol.78(2):256-265.)

In some aspects, the present disclosure is drawn to administeringmicrobial compositions described herein to modulate or shift themicrobiome of beef cattle.

In some embodiments, the microbiome is shifted through theadministration of one or more microbes to one or more sections of thegastrointestinal tract. In some embodiments, the microbiome is shiftedthrough the administration of one or more microbes to the rumen. Infurther embodiments, the one or more microbes are those selected fromTable 1 and/or Table 2. In some embodiments, the microbiome shift ormodulation includes a decrease or loss of specific microbes that werepresent prior to the administration of one or more microbes of thepresent disclosure. In some embodiments, the microbiome shift ormodulation includes an increase in microbes that were present prior tothe administration of one or more microbes of the present disclosure. Insome embodiments, the microbiome shift or modulation includes a gain ofone or more microbes that were not present prior to the administrationof one or more microbes of the present disclosure. In a furtherembodiment, the gain of one or more microbes is a microbe that was notspecifically included in the administered microbial composition.

In some embodiments, the administration of microbes of the presentdisclosure results in a sustained modulation of the microbiome such thatthe administered microbes are present in the microbiome for a period ofat least 1 to 10, 1 to 9, 1 to 8, 1 to 7, 1 to 6, 1 to 5, 1 to 4, 1 to3, 1 to 2, 2 to 10, 2 to 9, 2 to 8, 2 to 7, 2 to 6, 2 to 5, 2 to 4, 2 to3, 3 to 10, 3 to 9, 3 to 8, 3 to 7, 3 to 6, 3 to 5, 3 to 4, 4 to 10, 4to 9, 4 to 8, 4 to 7, 4 to 6, 4 to 5, 5 to 10, 5 to 9, 5 to 8, 5 to 7, 5to 6, 6 to 10, 6 to 9, 6 to 8, 6 to 7, 7 to 10, 7 to 9, 7 to 8, 8 to 10,8 to 9, 9 to 10, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 days.

In some embodiments, the administration of microbes of the presentdisclosure results in a sustained modulation of the microbiome such thatthe administered microbes are present in the microbiome for a period ofat least 1 to 10, 1 to 9, 1 to 8, 1 to 7, 1 to 6, 1 to 5, 1 to 4, 1 to3, 1 to 2, 2 to 10, 2 to 9, 2 to 8, 2 to 7, 2 to 6, 2 to 5, 2 to 4, 2 to3, 3 to 10, 3 to 9, 3 to 8, 3 to 7, 3 to 6, 3 to 5, 3 to 4, 4 to 10, 4to 9, 4 to 8, 4 to 7, 4 to 6, 4 to 5, 5 to 10, 5 to 9, 5 to 8, 5 to 7, 5to 6, 6 to 10, 6 to 9, 6 to 8, 6 to 7, 7 to 10, 7 to 9, 7 to 8, 8 to 10,8 to 9, 9 to 10, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 weeks.

In some embodiments, the administration of microbes of the presentdisclosure results in a sustained modulation of the microbiome such thatthe administered microbes are present in the microbiome for a period ofat least 1 to 10, 1 to 9, 1 to 8, 1 to 7, 1 to 6, 1 to 5, 1 to 4, 1 to3, 1 to 2, 2 to 10, 2 to 9, 2 to 8, 2 to 7, 2 to 6, 2 to 5, 2 to 4, 2 to3, 3 to 10, 3 to 9, 3 to 8, 3 to 7, 3 to 6, 3 to 5, 3 to 4, 4 to 10, 4to 9, 4 to 8, 4 to 7, 4 to 6, 4 to 5, 5 to 10, 5 to 9, 5 to 8, 5 to 7, 5to 6, 6 to 10, 6 to 9, 6 to 8, 6 to 7, 7 to 10, 7 to 9, 7 to 8, 8 to 10,8 to 9, 9 to 10, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 months.

In some embodiments, the presence of the administered microbes aredetected by sampling the gastrointestinal tract and using primers toamplify the 16S or 18S rDNA sequences, or the ITS rDNA sequences of theadministered microbes. In some embodiments, the administered microbesare one or more of those selected from Table 1 and/or Table 2. In someembodiments, the administered microbes are one or more of thosecomprising rDNA sequences selected from SEQ ID NO: 1-5993.

In some embodiments, the microbiome of beef cattle is measured byamplifying polynucleotides collected from gastrointestinal samples,wherein the polynucleotides may be 16S or 18S rDNA fragments, or ITSrDNA fragments of microbial rDNA. In one embodiment, the microbiome isfingerprinted by a method of denaturing gradient gel electrophoresis(DGGE) wherein the amplified rDNA fragments are sorted by where theydenature, and form a unique banding pattern in a gel that may be usedfor comparing the microbiome of the same beef cattle over time or themicrobiomes of multiple. In another embodiment, the microbiome isfingerprinted by a method of terminal restriction fragment lengthpolymorphism (T-RFLP), wherein labelled PCR fragments are digested usinga restriction enzyme and then sorted by size. In a further embodiment,the data collected from the T-RFLP method is evaluated by nonmetricmultidimensional scaling (nMDS) ordination and PERMANOVA statisticsidentify differences in microbiomes, thus allowing for theidentification and measurement of shifts in the microbiome. See alsoShanks et al. (2011. Appl. Environ. Microbiol. 77(9):2992-3001), Petriet al. (2013. PLOS one. 8(12):e83424), and Menezes et al. (2011. FEMSMicrobiol. Ecol. 78(2):256-265.)

In some embodiments, administration of one or more microbialcompositions results in a shift in the microbiome that increases thenumber and/or type of carbon dioxide fixing microbes. In someembodiments, administration of one or more microbial composition resultsin a shift in the microbiome that increases the number and/or type ofcarbon dioxide fixing microbes by at least 0.5%, at least 1%, at least5%, at least 10%, at least 15%, at least 20%, at least 25%, at least30%, at least 35%, at least 40%, at least 45%, at least 50%, at least55%, at least 60%, at least 65%, at least 70%, at least 75%, at least80%, at least 85%, at least 90%, at least 95%, at least 100%, at least200%, at least 300%, at least 400%, at least 500%, at least 600%, or atleast 700%. In some embodiments, administration of one or more microbialcomposition results in a shift in the microbiome that increases thenumber and/or type of carbon dioxide fixing microbes by at least about0.5%, at least about 1%, at least about 5%, at least about 10%, at leastabout 15%, at least about 20%, at least about 25%, at least about 30%,at least about 35%, at least about 40%, at least about 45%, at leastabout 50%, at least about 55%, at least about 60%, at least about 65%,at least about 70%, at least about 75%, at least about 80%, at leastabout 85%, at least about 90%, at least about 95%, at least about 100%,at least about 200%, at least about 300%, at least about 400%, at leastabout 500%, at least about 600%, or at least about 700%.

In some embodiments, administration of one or more microbialcompositions results in a shift in the microbiome that decreases thenumber and/or type of methanogenic microbes. In some embodiments,administration of one or more microbial composition results in a shiftin the microbiome that reduces the number and/or type of methanogenicmicrobes by at least 0.5%, at least 1%, at least 5%, at least 10%, atleast 15%, at least 20%, at least 25%, at least 30%, at least 35%, atleast 40%, at least 45%, at least 50%, at least 55%, at least 60%, atleast 65%, at least 70%, at least 75%, at least 80%, at least 85%, atleast 90%, or at least 95%. In some embodiments, administration of oneor more microbial composition results in a shift in the microbiome thatdecreases the number and/or type of methanogenic microbes by at leastabout 0.5%, at least about 1%, at least about 5%, at least about 10%, atleast about 15%, at least about 20%, at least about 25%, at least about30%, at least about 35%, at least about 40%, at least about 45%, atleast about 50%, at least about 55%, at least about 60%, at least about65%, at least about 70%, at least about 75%, at least about 80%, atleast about 85%, at least about 90%, or at least about 95%.

In some embodiments, administration of one or more microbialcompositions results in a shift in the microbiome that decreases thenumber and/or type of lactate producing microbes. In some embodiments,administration of one or more microbial composition results in a shiftin the microbiome that reduces the number and/or type of lactateproducing microbes by at least 0.5%, at least 1%, at least 5%, at least10%, at least 15%, at least 20%, at least 25%, at least 30%, at least35%, at least 40%, at least 45%, at least 50%, at least 55%, at least60%, at least 65%, at least 70%, at least 75%, at least 80%, at least85%, at least 90%, or at least 95%. In some embodiments, administrationof one or more microbial composition results in a shift in themicrobiome that decreases the number and/or type of lactate producingmicrobes by at least about 0.5%, at least about 1%, at least about 5%,at least about 10%, at least about 15%, at least about 20%, at leastabout 25%, at least about 30%, at least about 35%, at least about 40%,at least about 45%, at least about 50%, at least about 55%, at leastabout 60%, at least about 65%, at least about 70%, at least about 75%,at least about 80%, at least about 85%, at least about 90%, or at leastabout 95%.

In some embodiments, administration of one or more microbialcompositions results in a shift in the microbiome that increases thenumber and/or type of lactate degrading microbes. In some embodiments,administration of one or more microbial composition results in a shiftin the microbiome that increases the number and/or type of lactatedegrading microbes by at least 0.5%, at least 1%, at least 5%, at least10%, at least 15%, at least 20%, at least 25%, at least 30%, at least35%, at least 40%, at least 45%, at least 50%, at least 55%, at least60%, at least 65%, at least 70%, at least 75%, at least 80%, at least85%, at least 90%, at least 95%, at least 100%, at least 200%, at least300%, at least 400%, at least 500%, at least 600%, or at least 700%. Insome embodiments, administration of one or more microbial compositionresults in a shift in the microbiome that increases the number and/ortype of lactate degrading microbes by at least about 0.5%, at leastabout 1%, at least about 5%, at least about 10%, at least about 15%, atleast about 20%, at least about 25%, at least about 30%, at least about35%, at least about 40%, at least about 45%, at least about 50%, atleast about 55%, at least about 60%, at least about 65%, at least about70%, at least about 75%, at least about 80%, at least about 85%, atleast about 90%, at least about 95%, at least about 100%, at least about200%, at least about 300%, at least about 400%, at least about 500%, atleast about 600%, or at least about 700%.

In some embodiments, administration of one or more microbialcompositions results in a shift in the microbiome that increases thenumber and/or type of volatile fatty acid (VFA)-producing microbes. Insome embodiments, the VFAs include acetate, butyrate, propionate,isobutyrate, isovalerate, and valerate. In some embodiments,administration of one or more microbial composition results in a shiftin the microbiome that increases the number and/or type of VFA-producingmicrobes by at least 0.5%, at least 1%, at least 5%, at least 10%, atleast 15%, at least 20%, at least 25%, at least 30%, at least 35%, atleast 40%, at least 45%, at least 50%, at least 55%, at least 60%, atleast 65%, at least 70%, at least 75%, at least 80%, at least 85%, atleast 90%, at least 95%, at least 100%, at least 200%, at least 300%, atleast 400%, at least 500%, at least 600%, or at least 700%. In someembodiments, administration of one or more microbial composition resultsin a shift in the microbiome that increases the number and/or type ofVFA-producing microbes by at least about 0.5%, at least about 1%, atleast about 5%, at least about 10%, at least about 15%, at least about20%, at least about 25%, at least about 30%, at least about 35%, atleast about 40%, at least about 45%, at least about 50%, at least about55%, at least about 60%, at least about 65%, at least about 70%, atleast about 75%, at least about 80%, at least about 85%, at least about90%, at least about 95%, at least about 100%, at least about 200%, atleast about 300%, at least about 400%, at least about 500%, at leastabout 600%, or at least about 700%.

In some embodiments, administration of one or more microbialcompositions results in a shift in the microbiome that increases thenumber and/or type of microbes that are utilized as protein sources forthe animal. In some embodiments, administration of one or more microbialcomposition results in a shift in the microbiome that increases thenumber and/or type of microbes that are utilized as protein sources forthe animal by at least 0.5%, at least 1%, at least 5%, at least 10%, atleast 15%, at least 20%, at least 25%, at least 30%, at least 35%, atleast 40%, at least 45%, at least 50%, at least 55%, at least 60%, atleast 65%, at least 70%, at least 75%, at least 80%, at least 85%, atleast 90%, at least 95%, at least 100%, at least 200%, at least 300%, atleast 400%, at least 500%, at least 600%, or at least 700%. In someembodiments, administration of one or more microbial composition resultsin a shift in the microbiome that increases the number and/or type ofmicrobes that are utilized as protein sources for the animal by at leastabout 0.5%, at least about 1%, at least about 5%, at least about 10%, atleast about 15%, at least about 20%, at least about 25%, at least about30%, at least about 35%, at least about 40%, at least about 45%, atleast about 50%, at least about 55%, at least about 60%, at least about65%, at least about 70%, at least about 75%, at least about 80%, atleast about 85%, at least about 90%, at least about 95%, at least about100%, at least about 200%, at least about 300%, at least about 400%, atleast about 500%, at least about 600%, or at least about 700%.

In some embodiments, administration of one or more microbialcompositions results in a shift in the microbiome that increases thenumber and/or type of vitamin synthesizing microbes. In someembodiments, administration of one or more microbial composition resultsin a shift in the microbiome that increases the number and/or type ofvitamin synthesizing microbes by at least 0.5%, at least 1%, at least5%, at least 10%, at least 15%, at least 20%, at least 25%, at least30%, at least 35%, at least 40%, at least 45%, at least 50%, at least55%, at least 60%, at least 65%, at least 70%, at least 75%, at least80%, at least 85%, at least 90%, at least 95%, at least 100%, at least200%, at least 300%, at least 400%, at least 500%, at least 600%, or atleast 700%. In some embodiments, administration of one or more microbialcomposition results in a shift in the microbiome that increases thenumber and/or type of vitamin synthesizing microbes by at least about0.5%, at least about 1%, at least about 5%, at least about 10%, at leastabout 15%, at least about 20%, at least about 25%, at least about 30%,at least about 35%, at least about 40%, at least about 45%, at leastabout 50%, at least about 55%, at least about 60%, at least about 65%,at least about 70%, at least about 75%, at least about 80%, at leastabout 85%, at least about 90%, at least about 95%, at least about 100%,at least about 200%, at least about 300%, at least about 400%, at leastabout 500%, at least about 600%, or at least about 700%.

In some embodiments, administration of one or more microbialcompositions results in a shift in the microbiome that reduces theoverall alpha diversity of the microbial community. In some embodiments,administration of one or more microbial composition results in a shiftin the microbiome that reduces the overall alpha diversity of themicrobial community by at least 0.5%, at least 1%, at least 5%, at least10%, at least 15%, at least 20%, at least 25%, at least 30%, at least35%, at least 40%, at least 45%, at least 50%, at least 55%, at least60%, at least 65%, at least 70%, at least 75%, at least 80%, at least85%, at least 90%, at least 95%, at least 100%, at least 200%, at least300%, at least 400%, at least 500%, at least 600%, or at least 700%. Insome embodiments, administration of one or more microbial compositionresults in a shift in the microbiome that reduces the overall alphadiversity of the microbial community by at least about 0.5%, at leastabout 1%, at least about 5%, at least about 10%, at least about 15%, atleast about 20%, at least about 25%, at least about 30%, at least about35%, at least about 40%, at least about 45%, at least about 50%, atleast about 55%, at least about 60%, at least about 65%, at least about70%, at least about 75%, at least about 80%, at least about 85%, atleast about 90%, at least about 95%, at least about 100%, at least about200%, at least about 300%, at least about 400%, at least about 500%, atleast about 600%, or at least about 700%.

In some embodiments, the administration of microbes of the presentdisclosure results in a modulation or shift of the microbiome whichfurther results in a desired phenotype or improved trait.

Cattle Microbial Compositional Diversity

Bovine in a commercial settings have been found to exhibit a high degreeof animal-to-animal variability in terms of the microbial diversity ofthe rumen. The increased variability of the microbial compositions ofthe rumen may lead to a lower ability to reach a stable microbialcomposition. Lower variability in turn results in a considerabledifference in health, weight, and other attributes that affectcommercial viability of the animal. See Shabat S K B et al. (ISME J10:2958-2972.)

In some embodiments, the administration of one or more microbes and/orbioensembles of the present disclosure during feed transition in beefcattle decreases the variability of the rumen microbiome in cattle andfurther establishes a stable rumen microbiome.

In some embodiments, the variability of the rumen microbiome is measuredas the total number of species present in the rumen at one or morelocations.

In some embodiments, the administration of one or more microbes and/orbioensembles of the present disclosure reduces the amount of timerequired for the rumen microbiome to reach a stabilized state.

In some embodiments, the administration of one or more microbes and/orbioensembles of the present disclosure results in beef cattle of thepresent disclosure reaching a stabilized state of the rumen microbiome;a reduction in the variability of the rumen microbiome.

In some embodiments, the stabilized state of the rumen microbiome isreached when the rumen microbiome of beef cattle contains about 10,about 20, about, 30, about 40, about 50, about 60, about 70, about 80,about 90, about 100, about 120, about 130, about 140, about 150, about160, about 170, about 180, about 190, about 200, about 250, about 300,about 400, about 500, about 600, about 700, about 800, about 900, about1,000, about 1,500, about 2,000, about 2,500, about 3,000, about 3,500,about 4,000, about 4,500, about 5,000, about 5,500, about 6,000, about6,500, about 7,000, about 7,500, about 8,000, about 8,500, about 9,000,about 9,500, or about 10,000 different species.

In some embodiments, the stabilized state of the rumen microbiome isreached when the rumen microbiome of beef cattle contains between about10 to about 50, about 10 to about 100, about 50 to about 100, about 50to about 200, about 100 to about 150, about 100 to about 200, about 100to about 400, about 200 to about 500, about 200 to about 700, about 400to about 800, about 500 to about 1,000, about 500 to about 2,000, about1,000 to about 2,000, about 1,000 to about 5,000, about 5,000 to about7,000, about 5,000 to about 10,000, or about 8,000 to about 10,000different species.

In some embodiments, at least 5%, at least 10%, at least 20%, at least30%, at least 40%, at least 50%, at least 60%, at least 70%, at least80%, at least 90%, or at least 95% of the beef cattle in a feedtransition reach a stabilized state after administration of one or moremicrobes and/or bioensembles of the present disclosure.

MIC Scoring

According to the methods provided herein, a sample is processed todetect the presence of one or more microorganism types in the sample(FIG. 1, 1001 ; FIG. 2, 2001 ). The absolute number of one or moremicroorganism organism type in the sample is determined (FIG. 1, 1002 ;

FIG. 2, 2002 ). The determination of the presence of the one or moreorganism types and the absolute number of at least one organism type canbe conducted in parallel or serially. For example, in the case of asample comprising a microbial community comprising bacteria (i.e., onemicroorganism type) and fungi (i.e., a second microorganism type), theuser in one embodiment detects the presence of one or both of theorganism types in the sample (FIG. 1, 1001 ; FIG. 2, 2001 ). The user,in a further embodiment, determines the absolute number of at least oneorganism type in the sample—in the case of this example, the number ofbacteria, fungi or combination thereof, in the sample (FIG. 1, 1002 ;FIG. 2, 2002 ).

In one embodiment, the sample, or a portion thereof is subjected to flowcytometry (FC) analysis to detect the presence and/or number of one ormore microorganism types (FIG. 1, 1001, 1002 ; FIG. 2, 2001, 2002 ). Inone flow cytometer embodiment, individual microbial cells pass throughan illumination zone, at a rate of at least about 300*s⁻¹, or at leastabout 500*s⁻¹, or at least about 1000*s⁻¹. However, one of ordinaryskill in the art will recognize that this rate can vary depending on thetype of instrument is employed. Detectors which are gated electronicallymeasure the magnitude of a pulse representing the extent of lightscattered. The magnitudes of these pulses are sorted electronically into“bins” or “channels,” permitting the display of histograms of the numberof cells possessing a certain quantitative property (e.g., cell stainingproperty, diameter, cell membrane) versus the channel number. Suchanalysis allows for the determination of the number of cells in each“bin” which in embodiments described herein is an “microorganism type”bin, e.g., a bacteria, fungi, nematode, protozoan, archaea, algae,dinoflagellate, virus, viroid, etc.

In one embodiment, a sample is stained with one or more fluorescent dyeswherein a fluorescent dye is specific to a particular microorganismtype, to enable detection via a flow cytometer or some other detectionand quantification method that harnesses fluorescence, such asfluorescence microscopy. The method can provide quantification of thenumber of cells and/or cell volume of a given organism type in a sample.In a further embodiment, as described herein, flow cytometry isharnessed to determine the presence and quantity of a unique firstmarker and/or unique second marker of the organism type, such as enzymeexpression, cell surface protein expression, etc. Two- or three-variablehistograms or contour plots of, for example, light scattering versusfluorescence from a cell membrane stain (versus fluorescence from aprotein stain or DNA stain) may also be generated, and thus animpression may be gained of the distribution of a variety of propertiesof interest among the cells in the population as a whole. A number ofdisplays of such multiparameter flow cytometric data are in common useand are amenable for use with the methods described herein.

In one embodiment of processing the sample to detect the presence andnumber of one or more microorganism types, a microscopy assay isemployed (FIG. 1, 1001, 1002 ). In one embodiment, the microscopy isoptical microscopy, where visible light and a system of lenses are usedto magnify images of small samples. Digital images can be captured by acharge-couple device (CCD) camera. Other microscopic techniques include,but are not limited to, scanning electron microscopy and transmissionelectron microscopy. Microorganism types are visualized and quantifiedaccording to the aspects provided herein.

In another embodiment of the disclosure, in order to detect the presenceand number of one or more microorganism types, each sample, or a portionthereof is subjected to fluorescence microscopy. Different fluorescentdyes can be used to directly stain cells in samples and to quantifytotal cell counts using an epifluorescence microscope as well as flowcytometry, described above. Useful dyes to quantify microorganismsinclude but are not limited to acridine orange (AO), 4,6-di-amino-2phenylindole (DAPI) and 5-cyano-2,3 Dytolyl Tetrazolium Chloride (CTC).Viable cells can be estimated by a viability staining method such as theLIVE/DEAD® Bacterial Viability Kit (Bac-Light™) which contains twonucleic acid stains: the green-fluorescent SYTO 9™ dye penetrates allmembranes and the red-fluorescent propidium iodide (PI) dye penetratescells with damaged membranes. Therefore, cells with compromisedmembranes will stain red, whereas cells with undamaged membranes willstain green. Fluorescent in situ hybridization (FISH) extendsepifluorescence microscopy, allowing for the fast detection andenumeration of specific organisms. FISH uses fluorescent labelledoligonucleotides probes (usually 15-25 basepairs) which bindspecifically to organism DNA in the sample, allowing the visualizationof the cells using an epifluorescence or confocal laser scanningmicroscope (CLSM). Catalyzed reporter deposition fluorescence in situhybridization (CARD-FISH) improves upon the FISH method by usingoligonucleotide probes labelled with a horse radish peroxidase (HRP) toamplify the intensity of the signal obtained from the microorganismsbeing studied. FISH can be combined with other techniques tocharacterize microorganism communities. One combined technique is highaffinity peptide nucleic acid (PNA)-FISH, where the probe has anenhanced capability to penetrate through the Extracellular PolymericSubstance (EPS) matrix. Another example is LIVE/DEAD-FISH which combinesthe cell viability kit with FISH and has been used to assess theefficiency of disinfection in drinking water distribution systems.

In another embodiment, each sample, or a portion thereof is subjected toRaman micro-spectroscopy in order to determine the presence of amicroorganism type and the absolute number of at least one microorganismtype (FIG. 1, 1001-1002 ; FIG. 2, 2001-2002 ). Raman micro-spectroscopyis a non-destructive and label-free technology capable of detecting andmeasuring a single cell Raman spectrum (SCRS). A typical SCRS providesan intrinsic biochemical “fingerprint” of a single cell. A SCRS containsrich information of the biomolecules within it, including nucleic acids,proteins, carbohydrates and lipids, which enables characterization ofdifferent cell species, physiological changes and cell phenotypes. Ramanmicroscopy examines the scattering of laser light by the chemical bondsof different cell biomarkers. A SCRS is a sum of the spectra of all thebiomolecules in one single cell, indicating a cell's phenotypic profile.Cellular phenotypes, as a consequence of gene expression, usuallyreflect genotypes. Thus, under identical growth conditions, differentmicroorganism types give distinct SCRS corresponding to differences intheir genotypes and can thus be identified by their Raman spectra.

In yet another embodiment, the sample, or a portion thereof is subjectedto centrifugation in order to determine the presence of a microorganismtype and the number of at least one microorganism type (FIG. 1,1001-1002 ; FIG. 2, 2001-2002 ). This process sediments a heterogeneousmixture by using the centrifugal force created by a centrifuge. Moredense components of the mixture migrate away from the axis of thecentrifuge, while less dense components of the mixture migrate towardsthe axis. Centrifugation can allow fractionation of samples intocytoplasmic, membrane and extracellular portions. It can also be used todetermine localization information for biological molecules of interest.Additionally, centrifugation can be used to fractionate total microbialcommunity DNA. Different prokaryotic groups differ in theirguanine-plus-cytosine (G+C) content of DNA, so density-gradientcentrifugation based on G+C content is a method to differentiateorganism types and the number of cells associated with each type. Thetechnique generates a fractionated profile of the entire community DNAand indicates abundance of DNA as a function of G+C content. The totalcommunity DNA is physically separated into highly purified fractions,each representing a different G+C content that can be analyzed byadditional molecular techniques such as denaturing gradient gelelectrophoresis (DGGE)/amplified ribosomal DNA restriction analysis(ARDRA) (see discussion herein) to assess total microbial communitydiversity and the presence/quantity of one or more microorganism types.

In another embodiment, the sample, or a portion thereof is subjected tostaining in order to determine the presence of a microorganism type andthe number of at least one microorganism type (FIG. 1, 1001-1002 ; FIG.2, 2001-2002 ). Stains and dyes can be used to visualize biologicaltissues, cells or organelles within cells. Staining can be used inconjunction with microscopy, flow cytometry or gel electrophoresis tovisualize or mark cells or biological molecules that are unique todifferent microorganism types. In vivo staining is the process of dyeingliving tissues, whereas in vitro staining involves dyeing cells orstructures that have been removed from their biological context.Examples of specific staining techniques for use with the methodsdescribed herein include, but are not limited to: gram staining todetermine gram status of bacteria, endospore staining to identify thepresence of endospores, Ziehl-Neelsen staining, haematoxylin and eosinstaining to examine thin sections of tissue, papanicolaou staining toexamine cell samples from various bodily secretions, periodicacid-Schiff staining of carbohydrates, Masson's trichome employing athree-color staining protocol to distinguish cells from the surroundingconnective tissue, Romanowsky stains (or common variants that includeWright's stain, Jenner's stain, May-Grunwald stain, Leishman stain andGiemsa stain) to examine blood or bone marrow samples, silver stainingto reveal proteins and DNA, Sudan staining for lipids and Conklin'sstaining to detect true endospores. Common biological stains includeacridine orange for cell cycle determination; bismarck brown for acidmucins; carmine for glycogen; carmine alum for nuclei; Coomassie bluefor proteins; Cresyl violet for the acidic components of the neuronalcytoplasm; Crystal violet for cell walls; DAPI for nuclei; eosin forcytoplasmic material, cell membranes, some extracellular structures andred blood cells; ethidium bromide for DNA; acid fuchsine for collagen,smooth muscle or mitochondria; haematoxylin for nuclei; Hoechst stainsfor DNA; iodine for starch; malachite green for bacteria in the Gimenezstaining technique and for spores; methyl green for chromatin; methyleneblue for animal cells; neutral red for Nissl substance; Nile blue fornuclei; Nile red for lipohilic entities; osmium tetroxide for lipids;rhodamine is used in fluorescence microscopy; safranin for nuclei.Stains are also used in transmission electron microscopy to enhancecontrast and include phosphotungstic acid, osmium tetroxide, rutheniumtetroxide, ammonium molybdate, cadmium iodide, carbohydrazide, ferricchloride, hexamine, indium trichloride, lanthanum nitrate, lead acetate,lead citrate, lead(II) nitrate, periodic acid, phosphomolybdic acid,potassium ferricyanide, potassium ferrocyanide, ruthenium red, silvernitrate, silver proteinate, sodium chloroaurate, thallium nitrate,thiosemicarbazide, uranyl acetate, uranyl nitrate, and vanadyl sulfate.

In another embodiment, the sample, or a portion thereof is subjected tomass spectrometry (MS) in order to determine the presence of amicroorganism type and the number of at least one microorganism type(FIG. 1, 1001-1002 ; FIG. 2, 2001-2002 ). MS, as discussed below, canalso be used to detect the presence and expression of one or more uniquemarkers in a sample (FIG. 1, 1003-1004 ; FIG. 2, 2003-2004 ). MS is usedfor example, to detect the presence and quantity of protein and/orpeptide markers unique to microorganism types and therefore to providean assessment of the number of the respective microorganism type in thesample. Quantification can be either with stable isotope labelling orlabel-free. De novo sequencing of peptides can also occur directly fromMS/MS spectra or sequence tagging (produce a short tag that can bematched against a database). MS can also reveal post-translationalmodifications of proteins and identify metabolites. MS can be used inconjunction with chromatographic and other separation techniques (suchas gas chromatography, liquid chromatography, capillary electrophoresis,ion mobility) to enhance mass resolution and determination.

In another embodiment, the sample, or a portion thereof is subjected tolipid analysis in order to determine the presence of a microorganismtype and the number of at least one microorganism type (FIG. 1,1001-1002 ; FIG. 2, 2001-2002 ). Fatty acids are present in a relativelyconstant proportion of the cell biomass, and signature fatty acids existin microbial cells that can differentiate microorganism types within acommunity. In one embodiment, fatty acids are extracted bysaponification followed by derivatization to give the respective fattyacid methyl esters (FAMEs), which are then analyzed by gaschromatography. The FAME profile in one embodiment is then compared to areference FAME database to identify the fatty acids and theircorresponding microbial signatures by multivariate statistical analyses.

In the aspects of the methods provided herein, the number of uniquefirst makers in the sample, or portion thereof (e.g., sample aliquot) ismeasured, as well as the abundance of each of the unique first markers(FIG. 1, 1003 ; FIG. 2, 2003 ). A unique marker is a marker of amicroorganism strain. It should be understood by one of ordinary skillin the art that depending on the unique marker being probed for andmeasured, the entire sample need not be analyzed. For example, if theunique marker is unique to bacterial strains, then the fungal portion ofthe sample need not be analyzed. As described above, in someembodiments, measuring the absolute abundance of one or more organismtypes in a sample comprises separating the sample by organism type,e.g., via flow cytometry.

Any marker that is unique to an organism strain can be employed herein.For example, markers can include, but are not limited to, small subunitribosomal RNA genes (16S/18S rDNA), large subunit ribosomal RNA genes(23S/25S/28S rDNA), intercalary 5.8S gene, cytochrome c oxidase,beta-tubulin, elongation factor, RNA polymerase and internal transcribedspacer (ITS).

Ribosomal RNA genes (rDNA), especially the small subunit ribosomal RNAgenes, i.e., 18S rRNA genes (18S rDNA) in the case of eukaryotes and 16SrRNA (16S rDNA) in the case of prokaryotes, have been the predominanttarget for the assessment of organism types and strains in a microbialcommunity. However, the large subunit ribosomal RNA genes, 28S rDNAs,have been also targeted. rDNAs are suitable for taxonomic identificationbecause: (i) they are ubiquitous in all known organisms; (ii) theypossess both conserved and variable regions; (iii) there is anexponentially expanding database of their sequences available forcomparison. In community analysis of samples, the conserved regionsserve as annealing sites for the corresponding universal PCR and/orsequencing primers, whereas the variable regions can be used forphylogenetic differentiation. In addition, the high copy number of rDNAin the cells facilitates detection from environmental samples.

The internal transcribed spacer (ITS), located between the 18S rDNA and28S rDNA, has also been targeted. The ITS is transcribed but splicedaway before assembly of the ribosomes. The ITS region is composed of twohighly variable spacers, ITS1 and ITS2, and the intercalary 5.8S gene.This rDNA operon occurs in multiple copies in genomes. Because the ITSregion does not code for ribosome components, it is highly variable.

In one embodiment, the unique RNA marker can be an mRNA marker, an siRNAmarker or a ribosomal RNA marker.

Protein-coding functional genes can also be used herein as a uniquefirst marker. Such markers include but are not limited to: therecombinase A gene family (bacterial RecA, archaea RadA and RadB,eukaryotic Rad51 and Rad57, phage UvsX); RNA polymerase β subunit (RpoB)gene, which is responsible for transcription initiation and elongation;chaperonins. Candidate marker genes have also been identified forbacteria plus archaea: ribosomal protein S2 (rpsB), ribosomal proteinS10 (rpsJ), ribosomal protein L1 (rplA), translation elongation factorEF-2, translation initiation factor IF-2, metalloendopeptidase,ribosomal protein L22, ffh signal recognition particle protein,ribosomal protein L4/Lle (rp1D), ribosomal protein L2 (rp1B), ribosomalprotein S9 (rpsl), ribosomal protein L3 (rp1C), phenylalanyl-tRNAsynthetase beta subunit, ribosomal protein L14b/L23e (rp1N), ribosomalprotein S5, ribosomal protein S19 (rpsS), ribosomal protein S7,ribosomal protein L16/L10E (rp1P), ribosomal protein S13 (rpsM),phenylalanyl-tRNA synthetase α subunit, ribosomal protein L15, ribosomalprotein L25/L23, ribosomal protein L6 (rp1F), ribosomal protein L11(rp1K), ribosomal protein L5 (rplE), ribosomal protein S12/S23,ribosomal protein L29, ribosomal protein S3 (rpsC), ribosomal protein S11 (rpsK), ribosomal protein L10, ribosomal protein S8, tRNApseudouridine synthase B, ribosomal protein L18P/L5E, ribosomal proteinS15P/S13e, Porphobilinogen deaminase, ribosomal protein S17, ribosomalprotein L13 (rp1M), phosphoribosylformylglycinamidine cyclo-ligase(rpsE), ribonuclease HII and ribosomal protein L24. Other candidatemarker genes for bacteria include: transcription elongation protein NusA(nusA), rpoB DNA-directed RNA polymerase subunit beta (rpoB),GTP-binding protein EngA, rpoC DNA-directed RNA polymerase subunitbeta’, priA primosome assembly protein, transcription-repair couplingfactor, CTP synthase (pyrG), secY preprotein translocase subunit SecY,GTP-binding protein Obg/CgtA, DNA polymerase I, rpsF 30S ribosomalprotein S6, poA DNA-directed RNA polymerase subunit alpha, peptide chainrelease factor 1, rpll 505 ribosomal protein L9, polyribonucleotidenucleotidyltransferase, tsf elongation factor Ts (tsf), rplQ 50Sribosomal protein L17, tRNA (guanine-N(1)-)-methyltransferase (rp1S),rplY probable 50S ribosomal protein L25, DNA repair protein RadA,glucose-inhibited division protein A, ribosome-binding factor A, DNAmismatch repair protein MutL, smpB SsrA-binding protein (smpB),N-acetylglucosaminyl transferase, S-adenosyl-methyltransferase MraW,UDP-N-acetylmuramoylalanine-D-glutamate ligase, rpl S 50S ribosomalprotein L19, rp1T 50S ribosomal protein L20 (rp1T), ruvA Hollidayjunction DNA helicase, ruvB Holliday junction DNA helicase B, serSseryl-tRNA synthetase, rplU 50S ribosomal protein L21, rpsR 30Sribosomal protein S18, DNA mismatch repair protein MutS, rpsT 30Sribosomal protein S20, DNA repair protein RecN, frr ribosome recyclingfactor (frr), recombination protein RecR, protein of unknown functionUPF0054, miaA tRNA isopentenyltransferase, GTP-binding protein YchF,chromosomal replication initiator protein DnaA, dephospho-CoA kinase,16S rRNA processing protein RimM, ATP-cone domain protein,1-deoxy-D-xylulose 5-phosphate reductoisomerase, 2C-methyl-D-erythritol2,4-cyclodiphosphate synthase, fatty acid/phospholipid synthesis proteinPlsX, tRNA(Ile)-lysidine synthetase, dnaG DNA primase (dnaG), ruvCHolliday junction resolvase, rpsP 30S ribosomal protein S16, RecombinaseA recA, riboflavin biosynthesis protein RibF, glycyl-tRNA synthetasebeta subunit, trmU tRNA(5-methylaminomethyl-2-thiouridylate)-methyltransferase, rpml 50Sribosomal protein L35, hemE uroporphyrinogen decarboxylase, Rodshape-determining protein, rpmA 50S ribosomal protein L27 (rpmA),peptidyl-tRNA hydrolase, translation initiation factor IF-3 (infC),UDP-N-acetylmuramyl-tripeptide synthetase, rpmF 50S ribosomal proteinL32, rplL 50S ribosomal protein L7/L12 (rpIL), leuS leucyl-tRNAsynthetase, ligA NAD-dependent DNA ligase, cell division protein FtsA,GTP-binding protein TypA, ATP-dependent Clp protease, ATP-bindingsubunit ClpX, DNA replication and repair protein RecF andUDP-N-acetylenolpyruvoylglucosamine reductase.

Phospholipid fatty acids (PLFAs) may also be used as unique firstmarkers according to the methods described herein. Because PLFAs arerapidly synthesized during microbial growth, are not found in storagemolecules and degrade rapidly during cell death, it provides an accuratecensus of the current living community. All cells contain fatty acids(FAs) that can be extracted and esterified to form fatty acid methylesters (FAMEs). When the FAMEs are analyzed using gaschromatography—mass spectrometry, the resulting profile constitutes a‘fingerprint’ of the microorganisms in the sample. The chemicalcompositions of membranes for organisms in the domains Bacteria andEukarya are comprised of fatty acids linked to the glycerol by anester-type bond (phospholipid fatty acids (PLFAs)). In contrast, themembrane lipids of Archaea are composed of long and branchedhydrocarbons that are joined to glycerol by an ether-type bond(phospholipid ether lipids (PLELs)). This is one of the most widely usednon-genetic criteria to distinguish the three domains. In this context,the phospholipids derived from microbial cell membranes, characterizedby different acyl chains, are excellent signature molecules, becausesuch lipid structural diversity can be linked to specific microbialtaxa.

As provided herein, in order to determine whether an organism strain isactive, the level of expression of one or more unique second markers,which can be the same or different as the first marker, is measured(FIG. 1, 1004 ; FIG. 2, 2004 ). Unique first markers are describedabove. The unique second marker is a marker of microorganism activity.For example, in one embodiment, the mRNA or protein expression of any ofthe first markers described above is considered a unique second markerfor the purposes of this disclosure.

In one embodiment, if the level of expression of the second marker isabove a threshold level (e.g., a control level) or at a threshold level,the microorganism is considered to be active (FIG. 1, 1005 ; FIG. 2,2005 ). Activity is determined in one embodiment, if the level ofexpression of the second marker is altered by at least about 5%, atleast about 10%, at least about 15%, at least about 20%, at least about25%, or at least about 30%, as compared to a threshold level, which insome embodiments, is a control level.

Second unique markers are measured, in one embodiment, at the protein,RNA or metabolite level. A unique second marker is the same or differentas the first unique marker.

As provided above, a number of unique first markers and unique secondmarkers can be detected according to the methods described herein.Moreover, the detection and quantification of a unique first marker iscarried out according to methods known to those of ordinary skill in theart (FIG. 1, 1003-1004 , FIG. 2, 2003-2004 ).

Nucleic acid sequencing (e.g., gDNA, cDNA, rRNA, mRNA) in one embodimentis used to determine absolute cell count of a unique first marker and/orunique second marker. Sequencing platforms include, but are not limitedto, Sanger sequencing and high-throughput sequencing methods availablefrom Roche/454 Life Sciences, Illumina/Solexa, Pacific Biosciences, IonTorrent and Nanopore. The sequencing can be amplicon sequencing ofparticular DNA or RNA sequences or whole metagenome/transcriptomeshotgun sequencing.

Traditional Sanger sequencing (Sanger et al. (1977) DNA sequencing withchain-terminating inhibitors. Proc Natl. Acad. Sci. USA, 74, pp.5463-5467, incorporated by reference herein in its entirety) relies onthe selective incorporation of chain-terminating dideoxynucleotides byDNA polymerase during in vitro DNA replication and is amenable for usewith the methods described herein.

In another embodiment, the sample, or a portion thereof is subjected toextraction of nucleic acids, amplification of DNA of interest (such asthe rRNA gene) with suitable primers and the construction of clonelibraries using sequencing vectors. Selected clones are then sequencedby Sanger sequencing and the nucleotide sequence of the DNA of interestis retrieved, allowing calculation of the number of unique microorganismstrains in a sample.

454 pyrosequencing from Roche/454 Life Sciences yields long reads andcan be harnessed in the methods described herein (Margulies et al.(2005) Nature, 437, pp. 376-380; U.S. Pat. Nos. 6,274,320; 6,258,568;6,210,891, each of which is herein incorporated in its entirety for allpurposes). Nucleic acid to be sequenced (e.g., amplicons or nebulizedgenomic/metagenomic DNA) have specific adapters affixed on either end byPCR or by ligation. The DNA with adapters is fixed to tiny beads(ideally, one bead will have one DNA fragment) that are suspended in awater-in-oil emulsion. An emulsion PCR step is then performed to makemultiple copies of each DNA fragment, resulting in a set of beads inwhich each bead contains many cloned copies of the same DNA fragment.Each bead is then placed into a well of a fiber-optic chip that alsocontains enzymes necessary for the sequencing-by-synthesis reactions.The addition of bases (such as A, C, G, or T) trigger pyrophosphaterelease, which produces flashes of light that are recorded to infer thesequence of the DNA fragments in each well. About 1 million reads perrun with reads up to 1,000 bases in length can be achieved. Paired-endsequencing can be done, which produces pairs of reads, each of whichbegins at one end of a given DNA fragment. A molecular barcode can becreated and placed between the adapter sequence and the sequence ofinterest in multiplex reactions, allowing each sequence to be assignedto a sample bioinformatically.

Illumina/Solexa sequencing produces average read lengths of about 25basepairs (bp) to about 300 bp (Bennett et al. (2005) Pharmacogenomics,6:373-382; Lange et al. (2014). BMC Genomics 15, p. 63; Fadrosh et al.(2014) Microbiome 2, p. 6; Caporaso et al. (2012) ISME J, 6, p.1621-1624; Bentley et al. (2008) Accurate whole human genome sequencingusing reversible terminator chemistry. Nature, 456:53-59). Thissequencing technology is also sequencing-by-synthesis but employsreversible dye terminators and a flow cell with a field of oligosattached. DNA fragments to be sequenced have specific adapters on eitherend and are washed over a flow cell filled with specificoligonucleotides that hybridize to the ends of the fragments. Eachfragment is then replicated to make a cluster of identical fragments.Reversible dye-terminator nucleotides are then washed over the flow celland given time to attach. The excess nucleotides are washed away, theflow cell is imaged, and the reversible terminators can be removed sothat the process can repeat and nucleotides can continue to be added insubsequent cycles. Paired-end reads that are 300 bases in length eachcan be achieved. An Illumina platform can produce 4 billion fragments ina paired-end fashion with 125 bases for each read in a single run.Barcodes can also be used for sample multiplexing, but indexing primersare used.

The SOLiD (Sequencing by Oligonucleotide Ligation and Detection, LifeTechnologies) process is a “sequencing-by-ligation” approach, and can beused with the methods described herein for detecting the presence andabundance of a first marker and/or a second marker (FIG. 1, 1003-1004 ;FIG. 2, 2003-2004 ) (Peckham et al. SOLiD™ Sequencing and 2-BaseEncoding. San Diego, Calif.: American Society of Human Genetics, 2007;Mitra et al. (2013) Analysis of the intestinal microbiota using SOLiD16S rRNA gene sequencing and SOLiD shotgun sequencing. BMC Genomics,14(Suppl 5): S16; Mardis (2008) Next-generation DNA sequencing methods.Annu Rev Genomics Hum Genet, 9:387-402; each incorporated by referenceherein in its entirety). A library of DNA fragments is prepared from thesample to be sequenced, and are used to prepare clonal bead populations,where only one species of fragment will be present on the surface ofeach magnetic bead. The fragments attached to the magnetic beads willhave a universal P1 adapter sequence so that the starting sequence ofevery fragment is both known and identical. Primers hybridize to the P1adapter sequence within the library template. A set of fourfluorescently labelled di-base probes compete for ligation to thesequencing primer. Specificity of the di-base probe is achieved byinterrogating every 1st and 2nd base in each ligation reaction. Multiplecycles of ligation, detection and cleavage are performed with the numberof cycles determining the eventual read length. The SOLiD platform canproduce up to 3 billion reads per run with reads that are 75 bases long.Paired-end sequencing is available and can be used herein, but with thesecond read in the pair being only 35 bases long. Multiplexing ofsamples is possible through a system akin to the one used by Illumina,with a separate indexing run.

The Ion Torrent system, like 454 sequencing, is amenable for use withthe methods described herein for detecting the presence and abundance ofa first marker and/or a second marker (FIG. 1, 1003-1004 ; FIG. 2,2003-2004 ). It uses a plate of microwells containing beads to which DNAfragments are attached. It differs from all of the other systems,however, in the manner in which base incorporation is detected. When abase is added to a growing DNA strand, a proton is released, whichslightly alters the surrounding pH. Microdetectors sensitive to pH areassociated with the wells on the plate, and they record when thesechanges occur. The different bases (A, C, G, T) are washed sequentiallythrough the wells, allowing the sequence from each well to be inferred.The Ion Proton platform can produce up to 50 million reads per run thathave read lengths of 200 bases. The Personal Genome Machine platform haslonger reads at 400 bases. Bidirectional sequencing is available.Multiplexing is possible through the standard in-line molecular barcodesequencing.

Pacific Biosciences (PacBio) SMRT sequencing uses a single-molecule,real-time sequencing approach and in one embodiment, is used with themethods described herein for detecting the presence and abundance of afirst marker and/or a second marker (FIG. 1, 1003-1004 ; FIG. 2,2003-2004 ). The PacBio sequencing system involves no amplificationstep, setting it apart from the other major next-generation sequencingsystems. In one embodiment, the sequencing is performed on a chipcontaining many zero-mode waveguide (ZMW) detectors. DNA polymerases areattached to the ZMW detectors and phospholinked dye-labeled nucleotideincorporation is imaged in real time as DNA strands are synthesized. ThePacBio system yields very long read lengths (averaging around 4,600bases) and a very high number of reads per run (about 47,000). Thetypical “paired-end” approach is not used with PacBio, since reads aretypically long enough that fragments, through CCS, can be coveredmultiple times without having to sequence from each end independently.Multiplexing with PacBio does not involve an independent read, butrather follows the standard “in-line” barcoding model.

In one embodiment, where the first unique marker is the ITS genomicregion, automated ribosomal intergenic spacer analysis (ARISA) is usedin one embodiment to determine the number and identity of microorganismstrains in a sample (FIG. 1, 1003 , FIG. 2, 2003 ) (Ranjard et al.(2003). Environmental Microbiology 5, pp. 1111-1120, incorporated byreference in its entirety for all purposes). The ITS region hassignificant heterogeneity in both length and nucleotide sequence. Theuse of a fluorescence-labeled forward primer and an automatic DNAsequencer permits high resolution of separation and high throughput. Theinclusion of an internal standard in each sample provides accuracy insizing general fragments.

In another embodiment, fragment length polymorphism (RFLP) ofPCR-amplified rDNA fragments, otherwise known as amplified ribosomal DNArestriction analysis (ARDRA), is used to characterize unique firstmarkers and the abundance of the same in samples (FIG. 1, 1003 , FIG. 2,2003 ) (for additional detail, see Massol-Deya et al. (1995). Mol.Microb. Ecol. Manual. 3.3.2, pp. 1-18, the entirety of which is hereinincorporated by reference for all purposes). rDNA fragments aregenerated by PCR using general primers, digested with restrictionenzymes, electrophoresed in agarose or acrylamide gels, and stained withethidium bromide or silver nitrate.

One fingerprinting technique used in detecting the presence andabundance of a unique first marker is single-stranded-conformationpolymorphism (SSCP) (see Lee et al. (1996). Appl Environ Microbiol 62,pp. 3112-3120; Scheinert et al. (1996). J. Microbiol. Methods 26, pp.103-117; Schwieger and Tebbe (1998). Appl. Environ. Microbiol. 64, pp.4870-4876, each of which is incorporated by reference herein in itsentirety). In this technique, DNA fragments such as PCR productsobtained with primers specific for the 16S rRNA gene, are denatured anddirectly electrophoresed on a non-denaturing gel. Separation is based ondifferences in size and in the folded conformation of single-strandedDNA, which influences the electrophoretic mobility. Reannealing of DNAstrands during electrophoresis can be prevented by a number ofstrategies, including the use of one phosphorylated primer in the PCRfollowed by specific digestion of the phosphorylated strands with lambdaexonuclease and the use of one biotinylated primer to perform magneticseparation of one single strand after denaturation. To assess theidentity of the predominant populations in a given microbialcomposition, in one embodiment, bands are excised and sequenced, orSSCP-patterns can be hybridized with specific probes. Electrophoreticconditions, such as gel matrix, temperature, and addition of glycerol tothe gel, can influence the separation.

In addition to sequencing based methods, other methods for quantifyingexpression (e.g., gene, protein expression) of a second marker areamenable for use with the methods provided herein for determining thelevel of expression of one or more second markers (FIG. 1, 1004 ; FIG.2, 2004 ). For example, quantitative RT-PCR, microarray analysis, linearamplification techniques such as nucleic acid sequence basedamplification (NASBA) are all amenable for use with the methodsdescribed herein, and can be carried out according to methods known tothose of ordinary skill in the art.

In another embodiment, the sample, or a portion thereof is subjected toa quantitative polymerase chain reaction (PCR) for detecting thepresence and abundance of a first marker and/or a second marker (FIG. 1,1003-1004 ; FIG. 2, 2003-2004 ). Specific microorganism strains activityis measured by reverse transcription of transcribed ribosomal and/ormessenger RNA (rRNA and mRNA) into complementary DNA (cDNA), followed byPCR (RT-PCR).

In another embodiment, the sample, or a portion thereof is subjected toPCR-based fingerprinting techniques to detect the presence and abundanceof a first marker and/or a second marker (FIG. 1, 1003-1004 ; FIG. 2,2003-2004 ). PCR products can be separated by electrophoresis based onthe nucleotide composition. Sequence variation among the different DNAmolecules influences the melting behavior, and therefore molecules withdifferent sequences will stop migrating at different positions in thegel. Thus electrophoretic profiles can be defined by the position andthe relative intensity of different bands or peaks and can be translatedto numerical data for calculation of diversity indices. Bands can alsobe excised from the gel and subsequently sequenced to reveal thephylogenetic affiliation of the community members. Electrophoresismethods can include, but are not limited to: denaturing gradient gelelectrophoresis (DGGE), temperature gradient gel electrophoresis (TGGE),single-stranded-conformation polymorphism (SSCP), restriction fragmentlength polymorphism analysis (RFLP) or amplified ribosomal DNArestriction analysis (ARDRA), terminal restriction fragment lengthpolymorphism analysis (T-RFLP), automated ribosomal intergenic spaceranalysis (ARISA), randomly amplified polymorphic DNA (RAPD), DNAamplification fingerprinting (DAF) and Bb-PEG electrophoresis.

In another embodiment, the sample, or a portion thereof is subjected toa chip-based platform such as microarray or microfluidics to determinethe abundance of a unique first marker and/or presence/abundance of aunique second marker (FIG. 1, 1003-1004 , FIG. 2, 2003-2004 ). The PCRproducts are amplified from total DNA in the sample and directlyhybridized to known molecular probes affixed to microarrays. After thefluorescently labeled PCR amplicons are hybridized to the probes,positive signals are scored by the use of confocal laser scanningmicroscopy. The microarray technique allows samples to be rapidlyevaluated with replication, which is a significant advantage inmicrobial community analyses. In general the hybridization signalintensity on microarrays can be directly proportional to the abundanceof the target organism. The universal high-density 16S microarray (e.g.,PHYLOCHIP) contains about 30,000 probes of 16SrRNA gene targeted toseveral cultured microbial species and “candidate divisions”. Theseprobes target all 121 demarcated prokaryotic orders and allowsimultaneous detection of 8,741 bacterial and archaeal taxa. Anothermicroarray in use for profiling microbial communities is the FunctionalGene Array (FGA). Unlike PHYLOCHIPs, FGAs are designed primarily todetect specific metabolic groups of bacteria. Thus, FGA not only revealthe community structure, but they also shed light on the in situcommunity metabolic potential. FGA contain probes from genes with knownbiological functions, so they are useful in linking microbial communitycomposition to ecosystem functions. An FGA termed GEOCHIPcontains >24,000 probes from all known metabolic genes involved invarious biogeochemical, ecological, and environmental processes such asammonia oxidation, methane oxidation, and nitrogen fixation.

A protein expression assay, in one embodiment, is used with the methodsdescribed herein for determining the level of expression of one or moresecond markers (FIG. 1, 1004 ; FIG. 2, 2004 ). For example, in oneembodiment, mass spectrometry or an immunoassay such as an enzyme-linkedimmunosorbant assay (ELISA) is utilized to quantify the level ofexpression of one or more unique second markers, wherein the one or moreunique second markers is a protein.

In one embodiment, the sample, or a portion thereof is subjected toBromodeoxyuridine (BrdU) incorporation to determine the level of asecond unique marker (FIG. 1, 1004 ; FIG. 2, 2004 ). BrdU, a syntheticnucleoside analog of thymidine, can be incorporated into newlysynthesized DNA of replicating cells. Antibodies specific for BRdU canthen be used for detection of the base analog. Thus BrdU incorporationidentifies cells that are actively replicating their DNA, a measure ofactivity of a microorganism according to one embodiment of the methodsdescribed herein. BrdU incorporation can be used in combination withFISH to provide the identity and activity of targeted cells.

In one embodiment, the sample, or a portion thereof is subjected tomicroautoradiography (MAR) combined with FISH to determine the level ofa second unique marker (FIG. 1, 1004 ; FIG. 2, 2004 ). MAR-FISH is basedon the incorporation of radioactive substrate into cells, detection ofthe active cells using autoradiography and identification of the cellsusing FISH. The detection and identification of active cells atsingle-cell resolution is performed with a microscope. MAR-FISH providesinformation on total cells, probe targeted cells and the percentage ofcells that incorporate a given radiolabelled substance. The methodprovides an assessment of the in situ function of targetedmicroorganisms and is an effective approach to study the in vivophysiology of microorganisms. A technique developed for quantificationof cell-specific substrate uptake in combination with MAR-FISH is knownas quantitative MAR (QMAR).

In one embodiment, the sample, or a portion thereof is subjected tostable isotope Raman spectroscopy combined with FISH (Raman-FISH) todetermine the level of a second unique marker (FIG. 1, 1004 ; FIG. 2,2004 ). This technique combines stable isotope probing, Ramanspectroscopy and FISH to link metabolic processes with particularorganisms. The proportion of stable isotope incorporation by cellsaffects the light scatter, resulting in measurable peak shifts forlabelled cellular components, including protein and mRNA components.Raman spectroscopy can be used to identify whether a cell synthesizescompounds including, but not limited to: oil (such as alkanes), lipids(such as triacylglycerols (TAG)), specific proteins (such as hemeproteins, metalloproteins), cytochrome (such as P450, cytochrome c),chlorophyll, chromophores (such as pigments for light harvestingcarotenoids and rhodopsins), organic polymers (such aspolyhydroxyalkanoates (PHA), polyhydroxybutyrate (PHB)), hopanoids,steroids, starch, sulfide, sulfate and secondary metabolites (such asvitamin B12).

In one embodiment, the sample, or a portion thereof is subjected toDNA/RNA stable isotope probing (SIP) to determine the level of a secondunique marker (FIG. 1, 1004 ; FIG. 2, 2004 ). SIP enables determinationof the microbial diversity associated with specific metabolic pathwaysand has been generally applied to study microorganisms involved in theutilization of carbon and nitrogen compounds. The substrate of interestis labelled with stable isotopes (such as ¹³C or ¹⁵N) and added to thesample. Only microorganisms able to metabolize the substrate willincorporate it into their cells. Subsequently, ¹³C-DNA and ¹⁵N-DNA canbe isolated by density gradient centrifugation and used for metagenomicanalysis. RNA-based SIP can be a responsive biomarker for use in SIPstudies, since RNA itself is a reflection of cellular activity.

In one embodiment, the sample, or a portion thereof is subjected toisotope array to determine the level of a second unique marker (FIG. 1,1004 ; FIG. 2, 2004 ). Isotope arrays allow for functional andphylogenetic screening of active microbial communities in ahigh-throughput fashion. The technique uses a combination of SIP formonitoring the substrate uptake profiles and microarray technology fordetermining the taxonomic identities of active microbial communities.Samples are incubated with a ¹⁴C-labeled substrate, which during thecourse of growth becomes incorporated into microbial biomass. The¹⁴C-labeled rRNA is separated from unlabeled rRNA and then labeled withfluorochromes. Fluorescent labeled rRNA is hybridized to a phylogeneticmicroarray followed by scanning for radioactive and fluorescent signals.The technique thus allows simultaneous study of microbial communitycomposition and specific substrate consumption by metabolically activemicroorganisms of complex microbial communities.

In one embodiment, the sample, or a portion thereof is subjected to ametabolomics assay to determine the level of a second unique marker(FIG. 1, 1004 ; FIG. 2, 2004 ). Metabolomics studies the metabolomewhich represents the collection of all metabolites, the end products ofcellular processes, in a biological cell, tissue, organ or organism.This methodology can be used to monitor the presence of microorganismsand/or microbial mediated processes since it allows associating specificmetabolite profiles with different microorganisms. Profiles ofintracellular and extracellular metabolites associated with microbialactivity can be obtained using techniques such as gaschromatography-mass spectrometry (GC-MS). The complex mixture of ametabolomic sample can be separated by such techniques as gaschromatography, high performance liquid chromatography and capillaryelectrophoresis. Detection of metabolites can be by mass spectrometry,nuclear magnetic resonance (NMR) spectroscopy, ion-mobilityspectrometry, electrochemical detection (coupled to HPLC) and radiolabel(when combined with thin-layer chromatography).

According to the embodiments described herein, the presence andrespective number of one or more active microorganism strains in asample are determined (FIG. 1, 1006 ; FIG. 2, 2006 ). For example,strain identity information obtained from assaying the number andpresence of first markers is analyzed to determine how many occurrencesof a unique first marker are present, thereby representing a uniquemicroorganism strain (e.g., by counting the number of sequence reads ina sequencing assay). This value can be represented in one embodiment asa percentage of total sequence reads of the first maker to give apercentage of unique microorganism strains of a particular microorganismtype. In a further embodiment, this percentage is multiplied by thenumber of microorganism types (obtained at step 1002 or 2002, see FIG. 1and FIG. 2 ) to give the absolute abundance of the one or moremicroorganism strains in a sample and a given volume.

The one or more microorganism strains are considered active, asdescribed above, if the level of second unique marker expression is at athreshold level, higher than a threshold value, e.g., higher than atleast about 5%, at least about 10%, at least about 20% or at least about30% over a control level.

In another aspect of the disclosure, a method for determining theabsolute abundance of one or more microorganism strains is determined ina plurality of samples (FIG. 2 , see in particular, 2007). For amicroorganism strain to be classified as active, it need only be activein one of the samples. The samples can be taken over multiple timepoints from the same source, or can be from different environmentalsources (e.g., different animals).

The absolute abundance values over samples are used in one embodiment torelate the one or more active microorganism strains, with anenvironmental parameter (FIG. 2, 2008 ). In one embodiment, theenvironmental parameter is the presence of a second active microorganismstrain. Relating the one or more active microorganism strains to theenvironmental parameter, in one embodiment, is carried out bydetermining the co-occurrence of the strain and parameter by correlationor by network analysis.

In one embodiment, determining the co-occurrence of one or more activemicroorganism strains with an environmental parameter comprises anetwork and/or cluster analysis method to measure connectivity ofstrains or a strain with an environmental parameter within a network,wherein the network is a collection of two or more samples that share acommon or similar environmental parameter. In another embodiment, thenetwork and/or cluster analysis method may be applied to determining theco-occurrence of two or more active microorganism strains in a sample(FIG. 2, 2008 ). In another embodiment, the network analysis comprisesnonparametric approaches including mutual information to establishconnectivity between variables. In another embodiment, the networkanalysis comprises linkage analysis, modularity analysis, robustnessmeasures, betweenness measures, connectivity measures, transitivitymeasures, centrality measures or a combination thereof (FIG. 2, 2009 ).In another embodiment, the cluster analysis method comprises building aconnectivity model, subspace model, distribution model, density model,or a centroid model and/or using community detection algorithms such asthe Louvain, Bron-Kerbosch, Girvan-Newman, Clauset-Newman-Moore,Pons-Latapy, and Wakita-Tsurumi algorithms (FIG. 2, 2010 ).

In one embodiment, the cluster analysis method is a heuristic methodbased on modularity optimization. In a further embodiment, the clusteranalysis method is the Louvain method (See, e.g., the method describedby Blondel et al. (2008) Fast unfolding of communities in largenetworks. Journal of Statistical Mechanics: Theory and Experiment,Volume 2008, October 2008, incorporated by reference herein in itsentirety for all purposes).

In another embodiment, the network analysis comprises predictivemodeling of network through link mining and prediction, collectiveclassification, link-based clustering, relational similarity, or acombination thereof. In another embodiment, the network analysiscomprises differential equation based modeling of populations. Inanother embodiment, the network analysis comprises Lotka-Volterramodeling.

In one embodiment, relating the one or more active microorganism strainsto an environmental parameter (e.g., determining the co-occurrence) inthe sample comprises creating matrices populated with linkages denotingenvironmental parameter and microorganism strain associations.

In one embodiment, the multiple sample data obtained at step 2007 (e.g.,over two or more samples which can be collected at two or more timepoints where each time point corresponds to an individual sample) iscompiled. In a further embodiment, the number of cells of each of theone or more microorganism strains in each sample is stored in anassociation matrix (which can be in some embodiments, an abundancematrix). In one embodiment, the association matrix is used to identifyassociations between active microorganism strains in a specific timepoint sample using rule mining approaches weighted with association(e.g., abundance) data. Filters are applied in one embodiment to removeinsignificant rules.

In one embodiment, the absolute abundance of one or more, or two or moreactive microorganism strains is related to one or more environmentalparameters (FIG. 2, 2008 ), e.g., via co-occurrence determination.Environmental parameters are chosen by the user depending on thesample(s) to be analyzed and are not restricted by the methods describedherein. The environmental parameter can be a parameter of the sampleitself, e.g., pH, temperature, amount of protein in the sample.Alternatively, the environmental parameter is a parameter that affects achange in the identity of a microbial community (i.e., where the“identity” of a microbial community is characterized by the type ofmicroorganism strains and/or number of particular microorganism strainsin a community), or is affected by a change in the identity of amicrobial community. For example, an environmental parameter in oneembodiment, is the food intake of an animal. In one embodiment, theenvironmental parameter is the presence, activity and/or abundance of asecond microorganism strain in the microbial community, present in thesame sample.

In some embodiments described herein, an environmental parameter isreferred to as a metadata parameter.

Other examples of metadata parameters include but are not limited togenetic information from the host from which the sample was obtained(e.g., DNA mutation information), sample pH, sample temperature,expression of a particular protein or mRNA, nutrient conditions (e.g.,level and/or identity of one or more nutrients) of the surroundingenvironment/ecosystem), susceptibility or resistance to disease, onsetor progression of disease, susceptibility or resistance of the sample totoxins, efficacy of xenobiotic compounds (pharmaceutical drugs),biosynthesis of natural products, or a combination thereof.

For example, according to one embodiment, microorganism strain numberchanges are calculated over multiple samples according to the method ofFIG. 2 (i.e., at 2001-2007). Strain number changes of one or more activestrains over time is compiled (e.g., one or more strains that haveinitially been identified as active according to step 2006), and thedirectionality of change is noted (i.e., negative values denotingdecreases, positive values denoting increases). The number of cells overtime is represented as a network, with microorganism strainsrepresenting nodes and the abundance weighted rules representing edges.Markov chains and random walks are leveraged to determine connectivitybetween nodes and to define clusters. Clusters in one embodiment arefiltered using metadata in order to identify clusters associated withdesirable metadata (FIG. 2, 2008 ).

In a further embodiment, microorganism strains are ranked according toimportance by integrating cell number changes over time and strainspresent in target clusters, with the highest changes in cell numberranking the highest.

Network Analysis

Network and/or cluster analysis method in one embodiment, is used tomeasure connectivity of the one or more strains within a network,wherein the network is a collection of two or more samples that share acommon or similar environmental parameter. In one embodiment, networkanalysis comprises linkage analysis, modularity analysis, robustnessmeasures, betweenness measures, connectivity measures, transitivitymeasures, centrality measures or a combination thereof. In anotherembodiment, network analysis comprises predictive modeling of networkthrough link mining and prediction, social network theory, collectiveclassification, link-based clustering, relational similarity, or acombination thereof. In another embodiment, network analysis comprisesdifferential equation based modeling of populations. In yet anotherembodiment, network analysis comprises Lotka-Volterra modeling.

Cluster analysis method comprises building a connectivity model,subspace model, distribution model, density model, or a centroid model.

Network and cluster based analysis, for example, to carry out methodstep 2008 of FIG. 2 , can be carried out via a module. As used herein, acomponent and/or module can be, for example, any assembly, instructionsand/or set of operatively-coupled electrical components, and caninclude, for example, a memory, a processor, electrical traces, opticalconnectors, software (executing in hardware) and/or the like.

Cattle Pathogen Resistance and Clearance

In some aspects, the present disclosure is drawn to administering one ormore microbial compositions described herein to beef cattle to clear thegastrointestinal tract of pathogenic microbes. In some embodiments, thepresent disclosure is further drawn to administering microbialcompositions described herein to prevent colonization of pathogenicmicrobes in the gastrointestinal tract. In some embodiments, theadministration of microbial compositions described herein further clearpathogens from the integument and the respiratory tract of beef cattle,and/or prevent colonization of pathogens on the integument and in therespiratory tract. In some embodiments, the administration of microbialcompositions described herein reduce leaky gut/intestinal permeability,levels of histamine, production of lipopolysaccharides (LPS),inflammation, ketosis, laminitis, respiratory and metabolic acidosis,rumen acidosis, bloat, abomasal dysplasia, liver abscesses, and/orincidence of liver disease.

In some embodiments, the microbial compositions of the presentdisclosure comprise one or more microbes that are present in thegastrointestinal tract of beef cattle at a relative abundance of lessthan 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%,0.5%, 0.1%, or 0.01%.

In some embodiments, after administration of microbial compositions ofthe present disclosure the one or more microbes are present in thegastrointestinal tract of the beef cattle at a relative abundance of atleast 0.5%, 1%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%,60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95%.

Pathogenic microbes of beef cattle include the following: Clostridiumperfringens, Clostridium botulinum, Salmonella typi, Salmonellatyphimurium, Salmonella enterica, Salmonella pullorum, Erysipelothrixinsidiosa, Campylobacter jejuni, Campylobacter coli, Campylobacter lari,Listeria monocytogenes, Streptococcus agalactiae, Streptococcusdysgalactiae, Corynebacterium bovis, Mycoplasma sp., Citrobacter sp.,Enterobacter sp., Pseudomonas aeruginosa, Pasteurella sp., Bacilluscereus, Bacillus licheniformis, Streptococcus uberis, Staphylococcusaureus, and pathogenic strains of enteropathogenic, enteroinvasive, orenterohemorrhagic Escherichia coli, Staphylococcus aureus, Pasteurellamultocida, Mannheimia haemolytica, Histophilus somni, Mycoplasma bovis,and Aspergillus sp.

In some embodiments, the pathogenic microbes include viral pathogens. Insome embodiments, the pathogenic microbes are pathogenic to both beefcattle and humans. In some embodiments, the pathogenic microbes arepathogenic to either beef cattle or humans.

In some embodiments, the administration of compositions of the presentdisclosure to beef cattle modulate the makeup of the gastrointestinalmicrobiome such that the administered microbes outcompete microbialpathogens present in the gastrointestinal tract. In some embodiments,the administration of compositions of the present disclosure to beefcattle harboring microbial pathogens outcompetes the pathogens andclears the beef cattle of the pathogens. In some embodiments, theadministration of compositions of the present disclosure stimulate hostimmunity, and aids in clearance of the microbial pathogens. In someembodiments, the administration of compositions of the presentdisclosure introduce microbes that produce bacteriostatic and/orbactericidal components that decrease or clear the beef cattle of themicrobial pathogens. (U.S. Pat. No. 8,345,010).

In some embodiments, challenging beef cattle with a microbial colonizeror microbial pathogen after administering one or more compositions ofthe present disclosure prevents the microbial colonizer or microbialpathogen from growing to a relative abundance of greater than 15%, 14%,13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.5%, 0.1%, or0.01%. In further embodiments, challenging beef cattle with a microbialcolonizer or microbial pathogen after administering one or morecompositions of the present disclosure prevents the microbial colonizeror microbial pathogen from colonizing beef cattle.

In some embodiments, clearance of the microbial colonizer or microbialpathogen occurs occurs in less than 25 days, less than 24 days, lessthan 23 days, less than 22 days, less than 21 days, less than 20 days,less than 19 days, less than 18 days, less than 17 days, less than 16days, less than 15 days, less than 14 days, less than 13 days, less than12 days, less than 11 days, less than 10 days, less than 9 days, lessthan 8 days, less than 7 days, less than 6 days, less than 5 days, lessthan 4 days, less than 3 days, or less than 2 days post administrationof the one or more compositions of the present disclosure.

In some embodiments, clearance of the microbial colonizer or microbialpathogen occurs within 1-30 days, 1-25 days, 1-20 day, 1-15 days, 1-10days, 1-5 days, 5-30 days, 5-25 days, 5-20 days, 5-15 days, 5-10 days,10-30 days, 10-25 days, 10-20 days, 10-15 days, 15-30 days, 15-25 days,15-20 days, 20-30 days, 20-25 days, or 25-30 days post administration ofthe one or more compositions of the present disclosure.

Improved Traits

The rumen is a specialized stomach dedicated to the digestion of feedcomponents in ruminants. A diverse microbial population inhabits therumen, where their primary function revolves around converting thefibrous and non-fibrous carbohydrate components into useable sources ofenergy and protein (FIG. 3 ). Cellulose, in particular, forms up to 40%of plant biomass and is considered indigestible by mammals. It also istightly associated with other structural carbohydrates, includinghemicellulose, pectin, and lignin. The cellulolytic microbes in therumen leverage extensive enzymatic activity in order break thesemolecules down into simple sugars and volatile fatty acids. Thisenzymatic activity is critical to the extraction of energy from feed,and more efficient degradation ultimately provides more energy to theanimal. The soluble sugars found in the non-fibrous portion of the feedare also fermented into gases and volatile fatty acids such as butyrate,propionate, and acetate. Volatile fatty acids arising from the digestionof both the fibrous and non-fibrous components of feed are ultimatelythe main source of energy of the ruminant.

In some aspects, the present disclosure is drawn to administeringmicrobial compositions described herein to beef cattle to improve one ormore traits through the modulation of aspects of weight, musculature,digestive chemistry, efficiency of feed utilization and digestibility,fecal output, prevention of colonization of pathogenic microbes, andclearance of pathogenic microbes.

In some embodiments, the at least one improved trait is selected fromthe group consisting of: an increase in weight; an increase ofmusculature; an increase of fatty acid concentration in thegastrointestinal tract; an increase of fatty acid production in thegastrointestinal tract; an increase of fatty acid concentration in therumen; a decrease in lactate concentration in the rumen; an improvedefficiency in feed utilization and digestibility; an improved feedefficiency; an improved average daily weight gain; an increased finalbody weight; an improved dry matter intake; an increase inpolysaccharide and lignin degradation; an increase in fat, starch,and/or protein digestion; an increase in fatty acid concentration in therumen; pH balance in the rumen, an increase in vitamin availability; anincrease in mineral availability; an increase in amino acidavailability; an increase in milk production, a reduction in methaneand/or nitrous oxide emissions; a reduction in manure production; animproved efficiency of nitrogen utilization; an improved efficiency ofphosphorous utilization; an increased resistance to colonization ofpathogenic microbes that colonize cattle; reduced mortality; increasedproduction of antimicrobials; increased clearance of pathogenicmicrobes; increased resistance to colonization of pathogenic microbesthat colonize cattle; increased resistance to colonization of pathogenicmicrobes that infect humans; and any combination thereof; reducedincidence and/or prevalence of acidosis or bloat; reduced incidence ofabomasal dysplasia; reduced body temperature; reduction in theconcentration of CO₂ (dissolved or otherwise) in the rumen; increase inCO₂ fixation; reduction in microbial methanogenic populations; increasein CO₂ fixing microbes; increasing the concentration of B vitamins inthe rumen; an increase in mammalian and/or microbial synthesis ofvitamins; reducing alpha diversity of the microbiome residing in therumen; reducing histamine and LPS production; reducing leaky gut andpermeability of the gastrointestinal lining; reduction in respiratoryand metabolic acidosis; reduction in laminitis; reduction in ketosis;reduction of the incidence of liver disease and/or liver abscesses;reducing lactate concentrations in the rumen; increasing degradation oflactate in the rumen; increasing microbial lactate-degradingpopulations; wherein said increase or reduction is determined bycomparing against an animal not having been administered saidcomposition.

In some embodiments, the [CO₂] (dissolved or otherwise) is reduced by atleast 0.5%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%,15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 30%, 35%, 40%,45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% relativeto an animal not having been administered a composition of the presentdisclosure.

In some embodiments, the [CO₂] (dissolved or otherwise) in the rumen isreduced by at least 0.5%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%,12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%,30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or100% relative to an animal not having been administered a composition ofthe present disclosure.

In some embodiments, the ruminal pH is increased by at least 0.5%, 1%,2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%,18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 30%, 35%, 40%, 45%, 50%, 55%,60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% relative to an animalnot having been administered a composition of the present disclosure.

In some embodiments, the ruminal pH has an increased buffering capacityby at least 0.5%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%,13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 30%,35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100%relative to an animal not having been administered a composition of thepresent disclosure.

In some embodiments, the [carbonic acid] is reduced by at least 0.5%,1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%,17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 30%, 35%, 40%, 45%, 50%,55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% relative to ananimal not having been administered a composition of the presentdisclosure.

In some embodiments, the [carbonic acid] in the rumen is reduced by atleast 0.5%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%,15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 30%, 35%, 40%,45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% relativeto an animal not having been administered a composition of the presentdisclosure.

In some embodiments, the fecal output is reduced by at least 0.5%, 1%,2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%,18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 30%, 35%, 40%, 45%, 50%, 55%,60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% relative to an animalnot having been administered a composition of the present disclosure. Insome embodiments, the fecal output is reduced by less than 0.5%, 1%, 2%,3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%,19%, 20%, 21%, 22%, 23%, 24%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%,65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% relative to an animal nothaving been administered a composition of the present disclosure.

In some embodiments, the incidence of liver disease or liver abscessesis reduced by at least 0.5%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%,11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%,25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%,95%, or 100% relative to an animal not having been administered acomposition of the present disclosure.

In some embodiments, the incidence of bloat is reduced by at least 0.5%,1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%,17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 30%, 35%, 40%, 45%, 50%,55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% relative to ananimal not having been administered a composition of the presentdisclosure.

In some embodiments, the synthesis of one or more volatile fatty acidsis increased by at least 0.5%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%,11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%,25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%,95%, or 100% relative to an animal not having been administered acomposition of the present disclosure.

In some embodiments, the final body weight of the animals is increasedby at least 0.5%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%,13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 30%,35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100%relative to an animal not having been administered a composition of thepresent disclosure.

In some embodiments, the rate of weight gain of the animals is increasedby at least 0.5%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%,13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 30%,35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100%relative to an animal not having been administered a composition of thepresent disclosure.

In some embodiments, the lipopolysaccharide production in the animals isdecreased by at least 0.5%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%,11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%,25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%,95%, or 100% relative to an animal not having been administered acomposition of the present disclosure.

In some embodiments, improving the efficiency and digestibility ofanimal feed is desirable. In some embodiments, increasing thedegradation of lignocellulosic components from animal feed is desirable.Lignocellulosic components include lignin, cellulose, and hemicellulose.

In some embodiments, increasing the concentration of fatty acids in thegastrointestinal tract is desirable. Fatty acids include acetic acid,propionic acid, and butyric acid. In some embodiments, maintaining thepH balance in the gastrointestinal tract to prevent destruction ofbeneficial microbial compositions is desirable.

In some embodiments, decreasing the amount of methane and manureproduced by beef cattle is desirable

In some embodiments, a decrease in the amount of total manure producedis desirable. In further embodiments, a decrease in the total amount ofphosphorous and/or nitrogen in the total manure produced is desirable.

In some embodiments, improving the dry matter intake is desirable. Insome embodiments, improving the feed intake is desirable. In someembodiments, improving the efficiency of nitrogen utilization of thefeed and/or dry matter ingested by beef cattle is desirable.

In some embodiments, the improved traits of the present disclosure arethe result of the administration of the presently described microbialcompositions. It is thought that the microbial compositions modulate themicrobiome of beef cattle such that the biochemistry of the rumen ischanged in such a way that the gastrointestinal liquid and solidsubstratum are more efficiently and more completely degraded intosubcomponents and metabolites than the gastrointestinal tract of beefcattle not having been administered microbial compositions of thepresent disclosure.

In some embodiments, the increase in efficiency and the increase ofdegradation of the gastrointestinal substratum result in an increase inimproved traits of the present disclosure.

In some embodiments, the administration of one or more compositions ofthe present disclosure result in an improved feed efficiency of grainintensive and/or energy intensive diets. In some embodiments, theimproved feed efficiency measured as a decrease in the amount/volume offeces while maintaining or increasing the intake of the feed. In furtherembodiments, the grain intensive diet is that which contains 100%, 99%,98%, 97%, 96%, 95%, 94%, 93%, 92%, 91%, 90%, 89%, 88%, 87%, 86%, 85%,84%, 83%, 82%, 81%, 80%, 79%, 78%, 77%, 76%, 75%, 74%, 73%, 72%, 71%,70%, 69%, 68%, 67%, 65%, 64%, 63%, 62%, 61%, 60%, 59%, 58%, 57%, 56%,55%, 54%, 53%, 52%, 51%, 50%, 49%, 48%, 47%, 46%, 45%, 44%, 43%, 42%,41%, or 40% grains.

In some embodiments, the administration of one or more compositions ofthe present disclosure result in an improved feed efficiency in thepresence or absence of antibiotic agents.

In some embodiments, the administration of one or more compositions ofthe present disclosure result in an increase in the average daily weightgain of ruminants, as compared to those not having been administered theone or more compositions.

In some embodiments, the administration of one or more compositions ofthe present disclosure result in an increase in the dry matter intake ofruminants, as compared to those not having been administered the one ormore compositions.

In some embodiments, the administration of one or more compositions ofthe present disclosure result in a reduced incidence and/or prevalenceof acidosis or bloat in ruminants, as compared to those not having beenadministered the one or more compositions.

In some embodiments, the administration of one or more compositions ofthe present disclosure result in a reduced body temperature inruminants, as compared to those not having been administered the one ormore compositions. In further embodiments, the reduction in temperatureis at least 0.2° F., at least 0.4° F., at least 0.6° F., at least 0.8°F., at least 1° F., at least 1.2° F., at least 1.4° F., at least 1.6°F., at least 1.8° F., at least 2° F., at least 2.2° F., at least 2.4°F., at least 2.6° F., at least 2.8° F., at least 3° F., at least 3.2°F., at least 3.4° F., at least 3.6° F., at least 3.8° F., at least 4°F., at least 4.2° F., at least 4.4° F., at least 4.6° F., at least 4.8°F., at least 5° F., at least 5.2° F., at least 5.4° F., at least 5.6°F., at least 5.8° F., or at least 6° F.

In further embodiments, the reduction in temperature is at about 0.2°F., about 0.4° F., about 0.6° F., about 0.8° F., about 1° F., about 1.2°F., about 1.4° F., about 1.6° F., about 1.8° F., about 2° F., about 2.2°F., about 2.4° F., about 2.6° F., about 2.8° F., about 3° F., about 3.2°F., about 3.4° F., about 3.6° F., about 3.8° F., about 4° F., about 4.2°F., about 4.4° F., about 4.6° F., about 4.8° F., about 5° F., about 5.2°F., about 5.4° F., about 5.6° F., about 5.8° F., or about 6° F.

In some embodiments, the administration of one or more compositions ofthe present disclosure result in an increase in the quality grade of theresulting beef, as set forth by the USDA Beef Quality and Yield Grades.In further embodiments, the increase in the quality grade is an increaseor upgrade to USDA Prime, USDA Choice, or USDA Select quality grades, ascompared to those not having been administered the one or morecompositions. In some embodiments, the increase in the quality grade isan increase in the amount of meat per ruminant that is labelled as USDAPrime, USDA Choice, or USDA Select.

In some embodiments, the administration of one or more compositions ofthe present disclosure result in an increase in the amount of marbling(intramuscular fat) in the resulting meat of the ruminants. In furtherembodiments, the increase in the amount of marbling is an increase inmarbling grade to Prime⁺, Prime^(∘), Prime⁻, Choice⁺, Choice^(∘),Choice⁻, Select⁺, Select⁻, Standard⁺, Standard^(∘), Standard⁻, ascompared to those not having been administered the one or morecompositions.

In some embodiments, the administration of one of more compositions ofthe present disclosure result in an increase or decrease in the redcolor of the resulting meat from the ruminant. In some embodiments, theincrease in the red color of the meat is an increase to light cherry redto slightly dark red, moderately light red to moderately dark red,moderately dark red to dark red, dark red to very dark red, as comparedto those not having been administered the one or more compositions. Insome embodiments, the decrease in the red color of the meat is adecrease to light cherry red, light cherry red to slightly dark red,moderately light red to moderately dark red, or moderately dark red todark red, as compared to those not having been administered the one ormore compositions.

In some embodiments, the administration of one or more compositions ofthe present disclosure result in an increase or decrease in the textureof the resulting meat from the ruminant. In some embodiments, thedecrease in the texture is from coarse to slightly coarse, moderatelyfine, fine, or very fine, as compared to those not having beenadministered the one or more compositions. In some embodiments, theincrease in the texture is very fine, fine, moderately fine, slightlycoarse, or coarse.

In some embodiments, the administration of one or more compositions ofthe present disclosure result in an increase or decrease in theconcentration and/or amount of the following volatile components whichare known to modulate the flavor and/or aroma of the resulting meat fromthe ruminants: pentanal, hexanal, heptanal, nonanal, methional,12-methyltridecanal, nona-2(E)-enal, deca-2(E),4(E)-dienal, butanoicacid, hexanoic acid, delta-nonalactone, decan-2-one,3-hydroxy-2-butanone, 2,3-octanedione, 1-octene-3-ol, 2-pentyl furan,2-methyl-3-[methylthio]furan, 4-hydroxy-5-methyl-3(2H)-furanone (HMF),methylpyrazine,2,5-dimethylpyrazine,methylpyrazine,2,6-dimethylpyrazine, pyrazines, glycine, alanine,lysine, cysteine, methionine, glutamine, succinic acid, lactic acid,inosinic acid, orthophosphoric acid, pyrrolidone carboxylic acid,glucose, fructose, ribose, aspartic acid, histidine, asparagine,pyrrolidone carboxylic, carnosine, anserine, hypoxanthine, arginine,leucine, tryptophan, monosodium glutamate (MSG), inosine monophosphate(IMP), guanosine monophosphate (GMP), bis(2-methyl-3-furyl) disulfide,and 2-methyl-3-furanthiol.

In some embodiments, the increase of any one or more of the traits ofthe present disclosure is an increase of about 0.1%, about 0.2%, about0.3%, about 0.4%, about 0.5%, about 0.6%, about 0.7%, about 0.8%, about0.9%, about 1%, about 2%, about 3%, about 4%, about 5%, about 6%, about7%, about 8%, about 9%, about 10%, about 11%, about 12%, about 13%,about 14%, about 15%, about 16%, about 17%, about 18%, about 19%, about20%, about 21%, about 22%, about 23%, about 24%, about 25%, about 26%,about 27%, about 28%, about 29%, about 30%, about 31%, about 32%, about33%, about 34%, about 35%, about 36%, about 37%, about 38%, about 39%,about 40%, about 41%, about 42%, about 43%, about 44%, about 45%, about46%, about 47%, about 48%, about 49%, about 50%, about 51%, about 52%,about 53%, about 54%, about 55%, about 56%, about 57%, about 58%, about59%, about 60%, about 61%, about 62%, about 63%, about 64%, about 65%,about 66%, about 67%, about 68%, about 69%, about 70%, about 71%, about72%, about 73%, about 74%, about 75%, about 76%, about 77%, about 78%,about 79%, about 80%, about 81%, about 82%, about 83%, about 84%, about85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%,about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about98%, about 99%, or about 100% relative to an animal not having beenadministered one or more microbial compositions of the presentdisclosure.

In some embodiments, the increase of any one or more of the traits ofthe present disclosure is an increase of at least 0.1%, at least 0.2%,at least 0.3%, at least 0.4%, at least 0.5%, at least 0.6%, at least0.7%, at least 0.8%, at least 0.9%, at least 1%, at least 2%, at least3%, at least 4%, at least 5%, at least 6%, at least 7%, at least 8%, atleast 9%, at least 10%, at least 11%, at least 12%, at least 13%, atleast 14%, at least 15%, at least 16%, at least 17%, at least 18%, atleast 19%, at least 20%, at least 21%, at least 22%, at least 23%, atleast 24%, at least 25%, at least 26%, at least 27%, at least 28%, atleast 29%, at least 30%, at least 31%, at least 32%, at least 33%, atleast 34%, at least 35%, at least 36%, at least 37%, at least 38%, atleast 39%, at least 40%, at least 41%, at least 42%, at least 43%, atleast 44%, at least 45%, at least 46%, at least 47%, at least 48%, atleast 49%, at least 50%, at least 51%, at least 52%, at least 53%, atleast 54%, at least 55%, at least 56%, at least 57%, at least 58%, atleast 59%, at least 60%, at least 61%, at least 62%, at least 63%, atleast 64%, at least 65%, at least 66%, at least 67%, at least 68%, atleast 69%, at least 70%, at least 71%, at least 72%, at least 73%, atleast 74%, at least 75%, at least 76%, at least 77%, at least 78%, atleast 79%, at least 80%, at least 81%, at least 82%, at least 83%, atleast 84%, at least 85%, at least 86%, at least 87%, at least 88%, atleast 89%, at least 90%, at least 91%, at least 92%, at least 93%, atleast 94%, at least 95%, at least 96%, at least 97%, at least 98%, atleast 99%, or at least 100% relative to an animal not having beenadministered one or more microbial compositions of the presentdisclosure.

In some embodiments, the decrease of any one or more of the traits ofthe present disclosure is a decrease of about 0.1%, about 0.2%, about0.3%, about 0.4%, about 0.5%, about 0.6%, about 0.7%, about 0.8%, about0.9%, about 1%, about 2%, about 3%, about 4%, about 5%, about 6%, about7%, about 8%, about 9%, about 10%, about 11%, about 12%, about 13%,about 14%, about 15%, about 16%, about 17%, about 18%, about 19%, about20%, about 21%, about 22%, about 23%, about 24%, about 25%, about 26%,about 27%, about 28%, about 29%, about 30%, about 31%, about 32%, about33%, about 34%, about 35%, about 36%, about 37%, about 38%, about 39%,about 40%, about 41%, about 42%, about 43%, about 44%, about 45%, about46%, about 47%, about 48%, about 49%, about 50%, about 51%, about 52%,about 53%, about 54%, about 55%, about 56%, about 57%, about 58%, about59%, about 60%, about 61%, about 62%, about 63%, about 64%, about 65%,about 66%, about 67%, about 68%, about 69%, about 70%, about 71%, about72%, about 73%, about 74%, about 75%, about 76%, about 77%, about 78%,about 79%, about 80%, about 81%, about 82%, about 83%, about 84%, about85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%,about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about98%, about 99%, or about 100% relative to an animal not having beenadministered one or more microbial compositions of the presentdisclosure.

In some embodiments, the decrease of any one or more of the traits ofthe present disclosure is a decrease of at least 0.1%, at least 0.2%, atleast 0.3%, at least 0.4%, at least 0.5%, at least 0.6%, at least 0.7%,at least 0.8%, at least 0.9%, at least 1%, at least 2%, at least 3%, atleast 4%, at least 5%, at least 6%, at least 7%, at least 8%, at least9%, at least 10%, at least 11%, at least 12%, at least 13%, at least14%, at least 15%, at least 16%, at least 17%, at least 18%, at least19%, at least 20%, at least 21%, at least 22%, at least 23%, at least24%, at least 25%, at least 26%, at least 27%, at least 28%, at least29%, at least 30%, at least 31%, at least 32%, at least 33%, at least34%, at least 35%, at least 36%, at least 37%, at least 38%, at least39%, at least 40%, at least 41%, at least 42%, at least 43%, at least44%, at least 45%, at least 46%, at least 47%, at least 48%, at least49%, at least 50%, at least 51%, at least 52%, at least 53%, at least54%, at least 55%, at least 56%, at least 57%, at least 58%, at least59%, at least 60%, at least 61%, at least 62%, at least 63%, at least64%, at least 65%, at least 66%, at least 67%, at least 68%, at least69%, at least 70%, at least 71%, at least 72%, at least 73%, at least74%, at least 75%, at least 76%, at least 77%, at least 78%, at least79%, at least 80%, at least 81%, at least 82%, at least 83%, at least84%, at least 85%, at least 86%, at least 87%, at least 88%, at least89%, at least 90%, at least 91%, at least 92%, at least 93%, at least94%, at least 95%, at least 96%, at least 97%, at least 98%, at least99%, or at least 100% relative to an animal not having been administeredone or more microbial compositions of the present disclosure.

Network Analysis

A network and/or cluster analysis method, in one embodiment, is used tomeasure connectivity of the one or more strains within a network,wherein the network is a collection of two or more samples that share acommon or similar environmental parameter. In one embodiment, networkanalysis comprises linkage analysis, modularity analysis, robustnessmeasures, betweenness measures, connectivity measures, transitivitymeasures, centrality measures or a combination thereof. In anotherembodiment, network analysis comprises predictive modeling of networkthrough link mining and prediction, social network theory, collectiveclassification, link-based clustering, relational similarity, or acombination thereof. In another embodiment, network analysis comprisesmutual information, maximal information coefficient (MIC) calculations,or other nonparametric methods between variables to establishconnectivity. In another embodiment, network analysis comprisesdifferential equation based modeling of populations. In yet anotherembodiment, network analysis comprises Lotka-Volterra modeling.

The environmental parameter can be a parameter of the sample itself,e.g., pH, temperature, amount of protein in the sample. Alternatively,the environmental parameter is a parameter that affects a change in theidentity of a microbial community (i.e., where the “identity” of amicrobial community is characterized by the type of microorganismstrains and/or number of particular microorganism strains in acommunity), or is affected by a change in the identity of a microbialcommunity. For example, an environmental parameter in one embodiment, isthe food intake of an animal. In one embodiment, the environmentalparameter is the presence, activity and/or abundance of a secondmicroorganism strain in the microbial community, present in the samesample. In some embodiments, an environmental parameter is referred toas a metadata parameter.

Other examples of metadata parameters include but are not limited togenetic information from the host from which the sample was obtained(e.g., DNA mutation information), sample pH, sample temperature,expression of a particular protein or mRNA, nutrient conditions (e.g.,level and/or identity of one or more nutrients) of the surroundingenvironment/ecosystem), susceptibility or resistance to disease, onsetor progression of disease, susceptibility or resistance of the sample totoxins, efficacy of xenobiotic compounds (pharmaceutical drugs),biosynthesis of natural products, or a combination thereof.

Diagnostics

In some embodiments, a sample (serum, fecal, rumen, tissue, blood, etc.)is collected from a ruminant. In some embodiments, the sample is assayedfor the presence and/or quantity of one or more chemical substances. Insome embodiments the presence or absence of the one or more chemicalsubstances are diagnostic of a desirable trait described in the presentdisclosure.

In some embodiments, the one or more chemical substances may be selectedfrom pantothenate, homocysteine, glutamine, carnitine, D-gluconate,hypoxanthine, orotate, succinate, methylmalonate, aconitate,2-hydroxy-2-methyl succinate, allantoin, homocysteic acid, homocysteine,citrate, isocitrate, and cytosine.

In some embodiments, the increase of any one of the following chemicalsubstances in the blood or blood serum at greater than 0.1, 0.2, 0.3,0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7,1.8, 1.9, 2.0, 2.1, 2.2, 2.3. 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3.0, 3.1,3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9 or 4.0 fold is predictive of therumen bacterial community composition comprising bacteria from classFlavobacteriia.

In some embodiments, the increase of at least 0.7 fold of pantothenatein the blood or blood serum is predictive of the rumen bacterialcommunity composition comprising a higher proportion of bacteria fromclass Flavobacteriia.

In some embodiments, the increase of at least 0.7 fold of pantothenatein the blood or blood serum is predictive of the animal having a lowresidual feed intake (RFI), as compared to an animal without at least a0.7 fold increase of pantothenate.

In some embodiments, the present disclosure is drawn to a method ofdetermining the RFI of an animal. In some embodiments, the methodcomprises collecting a first sample of blood or blood serum at a firsttime point and collecting a second sample of blood or blood serum at asecond time point, assaying the samples for the presence andconcentration of pantothenate in the blood or blood serum, wherein theanimal is indicated to possess low RFI if the pantothenate exhibits afold increase of at least 0.7 between the first sample and the secondsample.

EXAMPLES Example I. Determination of Feed Efficiency in Beef Cattle

The objective of the study was to document and quantify the feedefficiency of steers fed finishing diets.

A total of 50 steers were adjusted to trial rations during a pretestadjustment period of at least 15 days. This initial adjustment periodenabled the animals to acclimate to the GROWSAFE (model 4000E, GrowSafeSystems Ltd., Airdrie, AB, Canada) feeding units and test rations.Steers were fed a step up diet for approximately 21 days beforereceiving a high concentrate finishing diet, as presented below.Rumensin was added to the finishing diet at a rate of 96 mg/hd/d. At theend of the first period, a 60 day trial was conducted with a minimum of50 steers for which RFI was determined using the GROWSAFE feed intakesystem. Residual feed intakes for steers was ranked together, andanimals were classified as low RFI (highly efficient), mid RFI, or highRFI (lowly efficient) by dividing them in thirds. From thisclassification, the low and high RFI steers were selected for furtherstudy.

TABLE 10 Finishing diet defined by crude protein (CP), total digestiblenutrients (TDN), and dry matter (DM). CP (%) TDN (%) % of diet (DM) Corn9.00 87.60 75.00 Sorghum Sudan Hay 8.33 54.00 15.00 44% CP Supplement44.00 73.00 10.00 Total Diet Nutrient Composition: 12.4% CP, 81.1% TDN.Rumensin was added at a rate of 96 mg/hd/d

Example II. Characterization of Microbial Communities of Steers thatDiffer in RFI

On day 1 and weekly throughout a 60 trial period, rumen content sampleswere collected via esophageal tubing. The rumen content was collectedwhile the steer was in a chute by passing the tubing through a Frickspeculum placed in the mouth and a rubber bulb was manipulated toprovide a suction once the tube was in the rumen to collect three rumensamples. A first sample consisted of 20 ml of rumen content. A secondsample consisted of rumen content that was added to a 15 ml conical tubeprefilled with stabilization solution and stored at 4° C. immediatelyafter. The conical tube was filled to the top with rumen content. Athird sample consisted of 20 ml of rumen content that was added to aconical tube prefilled with stop solution. After adding the rumencontent to the stop solution, the solution was mixed via inversionseveral times and stored at 4° C. immediately after. Samples 2 and 3were shipped to Ascus overnight on ice the day of or the day followingsample collection. In addition, up to 10 ml of venous blood wascollected directly after rumen content collection.

Immediately after rumen sampling, rumen pH was measured using anelectronic pH meter. Subsamples were frozen to stop microbialfermentation and stored at −20° C. for subsequent volatile fatty acids(acetate, propionate, butyrate, isobutyrate, and valerate) analyses.Ammonia-nitrogen (NH3-N) was analyzed according to the colorimetrictechnique described by Chaney and Marbach (1962. Clin. Chem.8(2):130-132). Flash frozen (liquid nitrogen and storage at −80° C.) andlive rumen content samples were utilized to determine the abundances ofbacterial species utilizing next-generation sequencing of the 16S rRNA,16S rDNA, and/or ITS sequences on the Illumina MiSeq sequencing platform(Illumina, Inc., San Diego, Calif.). Body weight, dry matter intake, andaverage daily gain for each animal was also measured at the time ofrumen sampling.

Example III. Dynamics Mediating Feed Efficiency in Cattle

Rumen microbes produce metabolites that are released into the rumenlumen and can be absorbed through the rumen epithelium or through theepithelium in the lower gastrointestinal tract. (See Hungate. The Rumenand Its Microbes, Elsevier. 1966.). The rumen microbes are responsiblefor the production of approximately 70% of the energy supply to theruminant, including production of organic acids such as acetate andpropionate. (See Seymour et al. Animal Feed Sci. Tech. 2005.119:155-169). Differences in the production of these metabolites as wellas variation in rate and quantity of absorption can contribute todivergences in nutrient utilization and efficiency of the ruminants, andmay lead to physiological or phenotypic changes. (See Huntington.Reproduction Nutrition Development. 1990. 30:35-47; Okine and Mathison.Journal of Animal Science. 1991. 69:3435-3445). However, it can bedifficult to distinguish the origin of many metabolites between those ofendogenous origin and metabolites of microbial origin. Althoughassociations between the rumen microbiome and physiological changes inthe host have been identified, it has yet to be determined themechanisms driving these changes and whether foundational, or keystone,species are responsible for the divergences in feed efficiency and otherphenotypes. (See Hungate. The Rumen Microbial Ecosystem. Annual Reviewof Ecology and Systematics. 1975. 39-66).

In order to address these critical knowledge gaps, a combination ofmicrobial genomics, metabolomics, and bioinformatics were utilized tofurther define variations in feed efficiency as determined by thedivergences in residual feed intake (RFI). Determination of the complexassociations and networks between the rumen microbiome, host metabolome,and differences in host phenotype can be facilitated by novelutilization of bioinformatics and machine learning to discoverphysiological patterns and identifying microbial factors. This exampleexamines the relationship among RFI, the rumen microbial community, andserum metabolome in order to identify potential biomarkers for feedefficiency in beef/feedlot cattle.

Fifty weaned steers of approximately 7 months of age were housed at thePlateau Research and Education Center in Crossville, Tenn. Animalsweighed 264±2.7 kg at the beginning of the study and transitioned to abackgrounding diet (11.57% crude protein and 76.93% total digestiblenutrients with 28 mg monensin/kg on a dry matter basis) for 14 daysprior to the start of the trial. Steers were adapted to the GROWSAFEsystem during that adaptation period. Body weight (BW) was measured at 7day intervals and daily feed intake measured using the GROWSAFE systemfor the length of the 70 day feed efficiency trial. Feed efficiency wasdetermined using RFI. (See Koch et al. Journal of Animal Science. 1963.22:486-494). At the conclusion of the trial, steers were ranked based onRFI. Low- or high-RFI was determined as 0.5 SD below or above the meanRFI, respectively

Approximately 9 mL of blood was sampled weekly via venipuncture from thecoccygeal vein into serum separator tubes (Corvac, Kendall Health Care,St. Louis, Mo.). Blood samples were centrifuged at 2,000×g for 20 min at4° C. Serum was decanted into 5 mL plastic culture tubes and stored at−80° C. for further analyses

Rumen samples were centrifuged at 4,000 rpm for 15 min, and the nucleicacids were isolated using the POWERVIRAL Environmental RNA/DNA IsolationKit (Mo Bio Laboratories, Inc., Carlsbad, Calif., USA). The 16S rRNAgene was amplified. (See Lane. 16S/23S rRNA sequencing. Nucleic acidtechniques in bacterial systematics. 1991; Muyzer et al. Applied andEnvironmental Microbiology. 1993. 59:695-700). Following amplification,PCR products were verified with a 2% agarose gel electrophoresis andpurified using AMPure XP bead (Beckman Coulter, Brea, Calif., USA). Thepurified amplicon library was quantified and sequenced on the MiSeqPlatform (Illumina, San Diego, Calif., USA) according to standardprotocols. (See Flores et al. Genome Biology. 2014. 15:531). Raw fastqreads were de-multiplexed on the MiSeq Platform (Illumina, San Diego,Calif., USA)

All raw sequencing data was trimmed of adapter sequences and phred33quality filtered using Trim Galore (See Krueger and Galore. A wrappertool around Cutadapt and FastQC to consistently apply quality andadapter trimming to FastQ files. 2015). 16S taxonomic sequenceclustering and classification was performed on filtered sequence datawith the RDP 16S rRNA database. (See Edgar. SINTAX: a simplenon-Bayesian taxonomy classifier for 16S amplicon reads. BioRxiv. 2016.074161; Edgar and Flyvbjerg. Bioinformatics. 2015. 31:3476-3482; andCole et al. Nucleic Acids Research. 2013. 42:D633-D642).

Serum samples (50 μL) from each steer were extracted for metabolomicanalysis using 0.1% formic acid in acetonitrile:water:methanol (2:2:1),as described previous. (See Kamphorst et al. Analytical Chemistry. 2011.83:9114-9122). Metabolites were separated using a Synergy Hydro-RPcolumn (100×2 mm, 2.5 μm particle size). Mobile phases consisted of A:97:3 H2O:MeOH with 11 mM tributylamine and 15 mM acetic acid and B:MeOH. The gradient consisted of the following: 0.0 min, 0% B; 2.5 min 0%B; 5.0 min, 20% B; 7.5 min, 20% B; 13 min, 55% B; 15.5 min, 95% B; 18.5min, 95% B; 19 min, 0% B, and 25 min, 0% B. Flow rate was set to aconstant 0.200 mL/min and the column temperature remained at 25° C. Theautosampler tray was maintained at 4° C. and 10 μL of sample wasinjected into the Dionex UltiMate 3000 UPLC system (Thermo FisherScientific, Waltham, Mass.). Electrospray ionization was used tointroduce the samples into an Exactive Plus Orbitrap MS (Thermo FisherScientific, Waltham, Mass.), using an established method. (See Kamphorstet al. Analytical Chemistry. 2011. 83:9114-9122; and Lu et al.Analytical Chemistry. 2010. 82:3212-3221).

Raw files obtained from Xcalibur MS software (Thermo Electron Corp.,Waltham, Mass.) were converted into the mzML format using ProteoWizard.(See Chambers et al. Nature Biotechnology. 2012. 30:918). The convertedfiles were imported into MAVEN (Metabolomic Analysis and VisualizationEngine for LC-MS Data), a software package. (See Clasquin et al. CurrentProtocols in Bioinformatics. 2012. 14.11.1-14.11.23). Peaks for theknown metabolites were picked in MAVEN, which automatically performsnon-linear retention time correction and calculates peak areas acrosssamples, using a preliminary mass error of ±20 ppm and retention timewindow of five min. The UTK Biological and Small Molecule MassSpectrometry Core (BSMMSC) has replicated and expanded the method ofRabinowitz and coworkers and final metabolite annotations were madeusing a library of 263 retention time-accurate m/z pairs taken from MS1spectra. (See Lu et al. Analytical Chemistry. 2010. 82:3212-3221). Theannotation parameters were verified previously with pure standards aspart of establishing the method. For a metabolite to be annotated as aknown compound, the eluted peak had to be found within two min of theexpected retention time, and the metabolite mass had to be within ±5 ppmof the expected value. Metabolite identities were confirmed using theMAVEN software package, and peak areas for each compound were integratedusing the Quan Browser function of the Xcalibur MS Software (ThermoElectron Corp., Waltham, Mass.).

Downstream analysis was performed in python. PCoA was performed onBray-Curtis distances and statistical significance was assessed throughAnalysis of Similarities (ANOSIM). (See Clarke. Austral Ecology. 1993.18:117-143). Alpha diversity was measured both in dominance andsingletons. (See Hammer et al. Palaeontol Electronica. 2001. 4:1-9).Regression based analysis was performed through Ordinary Least Squares(OLS) regression. (See Shi et al. The Annals of Applied Statistics.2016. 10:1019-1040). Feature selection and supervised machine learningwas performed on completed data through Random Forests. (See Breiman.Machine Learning. 2001. 45:5-32).

Other measurements of α-diversity, including equitability, Simpson'sEvenness E, Shannon's Diversity Index, and Observed OTU, were assessedfor normality using SAS 9.4 (SAS Institute, Cary, N.C.). All variableswere found to follow a non-normal distribution, and were analyzed usingWilcoxon Rank Sum and Kruskal Wallis test.

Alpha-diversity was measured by number of singletons, equitability,Simpson's Evenness, observed OTU, Good's coverage, chaol, and Shannon'sDiversity Index. With the exception of number of singletons, α-diversitymetrics did not differ between low- and high-RFI steers at the end ofthe study. Other α-diversity metrics did not differ between high- andlow-RFI steers, including equitability (p=0.24), Simpson's Evenness(p=0.19), Observed OTU (p=0.78), Good's coverage (p=0.14), chaol(p=0.78), and Shannon's Diversity Index (p=0.07). Phylogenetic diversityof the rumen bacterial communities also occurred over time with twodistinct communities arising (FIG. 10A and FIG. 10B).

A total of 109 known metabolites were identified. Residual feed intakewas predictive of serum metabolomic signature and rumen microbialcommunity signature at week 10 (FIG. 11A and FIG. 11B).Glucose-1-phosphate (p=0.03; FIG. 12 ) and glucose-6-phosphate (p=0.02;FIG. 12 ) differed between low- and high-RFI steers. Several other serummetabolites were predictive of rumen bacterial community structure,including pantothenate, aconitate, succinate,2-hydroxy-2-methylsuccinate, allantoin, homocysteic acid,citate/isocitrate, and cytosine (FIG. 13 ). Serum pantothenate abundancewas the greatest predictor of the rumen bacterial community composition,and was also found to be significantly different between low- andhigh-RFI steers (p=0.04; FIG. 13 ; FIG. 14A).

In addition to serum pantothenate abundance as a predictor of rumenbacterial community composition, panothenate abundances were alsoassociated with a class of rumen bacteria, Flavobacteriia.Flavobacteriia were predictive of pantothenate abundance in serum MeanFlavobacteriia abundance differed between steers with low and highpantothenate abundances (p=0.04; FIG. 14B).

While a number of singletons in the rumen bacteria differed between low-and high-RFI steers, other measurements of α-diversity did not differbetween the two groups. The data presented in this study suggest thatα-diversity may not be a significant contributing factor to feedefficiency phenotypes in stable microbial communities in growing beefsteers.

Glucose-1-phosphate and glucose-6-phosphate are both intermediatemetabolites of the pentose phosphate pathway in which G6P undergoesseveral enzymatic reactions to generate NADPH, and is most common inpathways involved in fatty acid and steroid production. (See Cori et al.Journal of Biological Chemistry. 1939. 129:629-639). Glucose-6-phosphatedehydrogenase, the first enzyme of the pentose phosphate pathway, is therate limiting enzyme of this pathway (See Laliotis et al. ComparativeBiochemistry and Physiology Part B: Biochemistry and Molecular Biology.2007. 147:627-634), which is estimated to supply 50 to 80% of the NADPHrequired to perform fatty acid synthesis in ruminants. (See Vernon.Lipid Metabolism in Ruminant Animals, Pergamon. 1981. P. 279-362; andBelk et al. Journal of Animal Science. 1993. 71:1796-1804). Accumulationof G6P and G1P in less efficient animals may indicate less enzymaticactivity or efficiency of the pentose phosphate pathway, which woulddecrease NADPH concentrations, resulting in less adipose accumulation.Both G1P and G6P in serum could potentially serve as biomarkers for feedefficiency, as increased concentrations are both are associated withdecreased feed efficiency.

The rumen microbiome produces several vital nutrients for the hostanimals, including organic acids that serve as glucogenic precursors, aswell as proteins and vitamins. (See Hungate. The Rumen and its Microbes.Elsevier. 1966). A nutrient produced by the rumen microbiota ispantothenate. Pantothenate plays a significant role in the metabolism offatty acids in ruminants and other species. (See Smith et al.Metabolism. 1987. 36:115-121; Palanker. Journal of Lipid Research. 2016.57:380-387). In this example, pantothenate was not only associated withgreater feed efficiency, but was also predictive of rumen bacterialcommunity composition. Pantothenate is a key component of coenzyme A(CoA), which is required to perform a variety of functions inintermediary metabolism of ruminants. (See Ragaller et al. Journal ofAnimal Physiology and Animal Nutrition. 2011. 95:6-16). Namely, CoA isresponsible for transfer of fatty acid components into and out of themitochondria. (See Ball. Vitamins in Foods. Analysis, Bioavailability,and Stability. Boca Raton: CRC Press. 2006). Pantothenate is produced byseveral species of bacteria in the rumen, and can then be released intothe rumen lumen to be absorbed by the host animal. One class of bacteriathat can generate pantothenate in the rumen are Flavobacteriia. It wasfound in this example that greater Flavobacteriia abundances wereassociated with greater pantothenate abundances, and Flavobacteriiaabundances were predictive of pantothenate quantities, supporting thatmore efficient steers may have greater abundances of Flavobacteriia,which may lead to increased abundance of pantothenate.

As with G1P and G6P, pantothenate can be identified through serum;however, whereas G1P and G6P may serve as indicators of lower feedefficiency, pantothenate may indicate greater feed efficiency. Therelationship between pantothenate and Flavobacteriia can not onlyprovide insight as to mechanisms accounting for some variability in feedefficiency, but also serve as biochemical and microbial biomarkers inthe serum and rumen, respectively. These biomarkers could allowproducers to identify and select animals of greater feed efficiency.Metabolites and microbes predictive of efficiency phenotypes in cattleare not only imperative to partially explaining divergences in feedefficiency, but also to the selection of microbial communities relatedto efficient animals. These insights may also lead to the ability toselect for an optimal rumen microbiome.

This example identified potential microbial and biochemical biomarkersthat were used to determine extremes in feed efficiency in steers.Although, notable correlations between G1P and G6P and feed efficiencywere identified, linking, and perhaps predicting, the functionalcapacity of the rumen and its microbiome, specifically Flavobacteriia,through serum pantothenate offers the potential to use serumbiochemistry as an indicator in identification of feed efficient cattle.Additionally, although it has yet to be determined to what degree therumen microbiome influences the host, or the host influences the rumenmicrobiome, the present example identified several key physiologicalelements that may impact or predict microbial community structure (e.g.RFI), or predictive of RFI (i.e. the serum metabolome and rumenmicrobial community).

Example IV. Dynamics Mediating Feed Efficiency in Cattle

The majority of research aimed at determining the influence of themicrobiome on cattle production or the influence of cattle production onthe ruminal microbiome has been conducted by examining short-term, endpoint sampling or single periods of sample collection. Thesesingle-point analyses have provided valuable insight to the influence ofthe rumen microbiome on livestock production but may confound theinterpretation of study results and conclusions. Variation of theruminal microbiota between animals is considerable (See Ross et al.2012. BMC genetics 13(1):53; and Myer et al. 2015. PLOS ONE 10(6):e0129174), end point samples may not be satisfactory to adequatelydefine an existing state within a population. Studies examining bovinenutrition with regard to the ruminal microbiome routinely rely ondifferences in diet or diet transitions, where the length of the studyis defined by traditional nutritional parameters and historical data,not taking into account microbial acclimation to the study ration. Theruminal microbial temporal stability following such perturbations hasyet to be determined. Thus, diet acclimation prior to experiments, dietre-acclimation among experimental periods, and other dietary changes incattle gut microbiome studies may confound microbial characterizationwhen temporal variation and stability are not taken into consideration.These patterns of variation may have significant implications whenaiming to determine relationships between the gut microbiome andnutrition in cattle.

Fifty steers of approximately seven months of were obtained. The steersweighed 264±2.7 kg at the beginning of the trial. The steers grazed oncool-season grasses until being transferred to the GROWSAFE system for a14d adaptation period. Animals were placed on a step-up diet during the14d adaptation period and transitioned to a growing diet (11.57% crudeprotein and 76.93% total digestible nutrients on a dry matter basis)with 28 mg monensin/kg DM. A 70d feed efficiency trial was administeredfollowing the acclimation period. Weekly, body weight (BW) via chutescale, rumen fluid samples via gastric tubing, and blood samples viacoccygeal venipuncture were collected. (See Krysl and Hess. 1993.Journal of Animal Science 71(9): 2546-2555). Approximately 100 mL ofrumen content were transferred to 50 mL conical tubes, rumen content pHdetermined, and samples stored at −80° C. Feed intake was continuallymonitored via the GROWSAFE system throughout the 70d feed efficiencytrial.

Rumen samples were centrifuged at 4,000 rpm for 15 min, were isolatedusing the POWERVIRAL Environmental RNA/DNA Isolation Kit (Mo BioLaboratories, Inc., Carlsbad, Calif., USA). The 16S rRNA gene wasamplified for Illumina sequencing. Following amplification, PCR productswere verified with a standard agarose gel electrophoresis and purifiedusing AMPure XP bead (Beckman Coulter, Brea, Calif., USA). The purifiedamplicon library was quantified and sequenced on the MiSeq Platform(Illumina, San Diego, Calif., USA) according to standard protocols(Flores, Caporaso et al. 2014). Raw fastq read were de-multiplexed onthe MiSeq Platform (Illumina, San Diego, Calif., USA).

All raw sequencing data was trimmed of adapter sequences and phred33quality filtered using Trim Galore (See Krueger and Galore. A wrappertool around Cutadapt and FastQC to consistently apply quality andadapter trimming to FastQ files. 2015). 16S taxonomic sequenceclustering and classification was performed on filtered sequencing datawith the RDP 16S rRNA database. (See Edgar. SINTAX: a simplenon-Bayesian taxonomy classifier for 16S amplicon reads. BioRxiv. 2016.074161; Edgar and Flyvbjerg. Bioinformatics. 2015. 31:3476-3482; andCole et al. Nucleic Acids Research. 2013. 42:D633-D642).

Downstream analysis was performed in python. PCoA was performed onBray-Curtis distances and statistical significance was assessed throughAnalysis of Similarities (ANOSIM). (See Clarke. Austral Ecology. 1993.18:117-143). Alpha diversity was measured both in dominance andsingletons. (See Hammer et al. Palaeontol Electronica. 2001. 4:1-9).Regression based analysis was performed through Ordinary Least Squares(OLS) regression. (See Shi et al. The Annals of Applied Statistics.2016. 10:1019-1040). Feature selection and supervised machine learningwas performed on completed data through Random Forests. (See Breiman.Machine Learning. 2001. 45:5-32).

Other measurements of α-diversity, including equitability, Simpson'sEvenness E, Shannon's Diversity Index, and Observed OTU, were assessedfor normality using SAS 9.4 using the PROC UNIVARIATE command (SASInstitute, Cary, N.C.). All variables were found to follow a non-normaldistribution, and were analyzed using Wilcoxon Rank Sum and KruskalWallis test.

Alpha-diversity was measured using, equitability, Simpson's Evenness,chaol, Shannon's Diversity Index, and observed OTU. Good's coverage wasalso measured to ensure satisfactory coverage of OTU for each week.Shannon's Diversity Index was greatest at the beginning of the trial(4.66±0.32), but decreased overall by the end of the trial (3.61±0.14;P<0.0001). Observed OTU also was greater during the first week(160.14±12.83) compared to the final week (57.9±2.5; P<0.0001).Equitability was greatest during week 1 of the trial (0.66±0.03),fluctuated throughout the trial, but was lower at week 10 (0.61±0.02;P<0.0001). Whereas several metrics related to richness increased fromweek 1 to week 10, overall evenness fluctuated greatly (P<0.0001) butwas numerically similar by the end of the trial (0.12±0.02) to the firstweek of the trial (0.13±0.01). Greater phylogenetic diversity wasobserved at week 5 compared to week 1, but decreased by week 10 asevidence by spatial co-clustering. The rumen microbial community beganto shift at week and reached stabilization by week 10. Three orders wereidentified as changing significantly during the shift to the finalmicrobial community, including Pasteurellales, Aeromonadales, andBacteriodales. The shift to the final bacterial community compositionbegan to occur at week 4 (FIG. 4 ). Several physiological factors werepredictive of the rumen microbial community. Rumen pH was correlatedwith α-diversity (P=0.005; FIG. 4 ) at week 5 and predictive of rumenmicrobial community signature at week 10 (FIG. 25 ).

The diversity of the rumen bacterial community was greatest at the startof the trial, following a field-standard two week adaptation period tothe growing diet and was greatly variable throughout much of the trial.Rumen bacterial community diversity was lowest by the end of the trial,at ten weeks following the adaptation period. The rumen bacterialdiversity was greatest at the start of the trial following theadaptation period, but reached stability by ten weeks. The transitionfrom a predominantly forage-based diet to a diet incorporatingconcentrates causes a shift in bacterial taxa due to changes insubstrate type (See Tajima et al. 2001. Applied and EnvironmentalMicrobiology 67(6): 2766-2774). These differences in nutrientavailability to the microbes may have resulted in a state of microbialcommunity dysbiosis as bacterial populations competed for nutritionalsources, increased functional redundancy occurred, and the physicalenvironment of the rumen, such as pH, changed (See Whittaker. 1972.Taxon 21(2/3): 213-251).

Dietary transitions, adaptation periods, and wash-out periods are oftenincorporated into nutritional studies, including those involving therumen microbiome adaptation and wash-out periods have historicallyspanned anywhere from several days to four weeks, which previous studieshave suggested is adequate time for acclimation, as reviewed by (SeeBrown et al. 2006. Journal of Animal Science 84(13_suppl): E25-E33) andsupported by recent nutritional microbial studies (See Anderson et al.2016. Journal of applied microbiology 120(3): 588-599). However, in thisstudy, it took approximately ten weeks for the rumen microbial communityto stabilize, a time frame much longer than traditionally utilized foradaptation or wash-out periods. Although cattle may physically acclimateto feed within a two week period, the results of this study suggest therumen microbiome requires additional time to stabilize.

At week 4 of this study, three orders appeared to drive the shift to astable bacterial community composition, including Aeromonadales,Pasteurellales, and Bacteroidales. Two of these orders, Aeromonadalesand Pasteurellales, belong to the phylum Proteobacteria, andBacteroidales belongs to the phylum Bacteroidetes. Proteobacteria andBacteroidetes are typically two of the three most prevalent bacterialphyla found in the rumen but bacteria belonging to the phylum Firmicutesare frequently found to be the most abundant in the rumen, followed byBacteroidetes and Proteobacteria in cattle on a primarily forage-baseddiet. However, as cattle transition to incorporate morereadily-fermentable feedstuffs, Bacteroidetes becomes the dominantphylum.

The changes in abundances of these orders also provides additionalinsight given their function in the rumen. At week 4, relative abundanceof Aeromonadales sharply decreased, whereas Bacteroidales andPasteurellales both increased at week 4. Some rumen microbes belongingto Aeromonadales, including Ruminobacter and Succinovibrio, arefibrolytic in nature and found in conjunction with high-fiber diets,which may account for the decrease in abundance of those microbes as therumen microbiome adapted to the more readily digestible diet. Generafound in Bacteroidales and Pasteurellales, including Prevotella andActinobacillus, respectively, are important for digestion of protein andcarbohydrates. Rapid production of byproducts of metabolism, such asorganic acids, by Bacteroidales and Pasteurellales may be responsiblefor the shift in pH seen in week 5, which was also indicative of rumenmicrobiome signature. The diet fed in this study included feedstuffsthat are more readily fermentable, which can cause decreases in rumen pHdue to increased production of organic acids such as lactate. This canshift the bacterial community composition towards those bacteria thatare more tolerant of low pH, including those in Bacteroidales andPasteurellales (See Fernando et al. 2010. Applied and EnvironmentalMicrobiology 76(22): 7482-7490).

Example V. Dynamics Mediating Efficiency with a High Corn Diet

The lactate and other acids produced in the rumen are generallyconsidered the main drivers of acidosis and feed inefficiency in highcorn or grain intensive diets, like those provided in beef/cattlefeedlots. Many in the art believe that higher concentrations of volatilefatty acids (VFAs) induce acidosis, and that lactate producers dominatethe rumen microbiome during acidosis, dropping the pH faster than otherfermentation acids. Many believe that bringing acidotic feedlot cattleback to a healthy state requires increasing the ruminal pH by reducinglactate and VFA concentration, or through the use of feed additives suchas buffers for pH stability or ionophores to inhibit lactate producers.

The table that occurs in FIG. 6 identifies the ruminal VFAconcentrations and pH of Blank Angus steers between high RFI and low RFIat week 10. The low RFI animals tended to have a higher production ofVFAs compared to the high RFI animals, and the pH is similar for bothgroups.

The pH is measured by taking the pK_(a) plus the log of HCO₃— divided bydissolved CO₂ (dCO₂). CO₂ is known to play a key role in respiration andblood buffering, and it likely plays a large role in pH regulation ofthe rumen. In the rumen, CO₂ is present in the rumen gas cap and therumen liquid. Most is in the rumen liquid as dCO₂ as a base(bicarbonate) or as an acid (carbonic acid). The CO₂ in the liquidtransforms into carbonic acid (˜1%), which quickly dissociates tobicarbonate, the primary base in the rumen fluid. (See Laporte-Uribe.2016. Animal Feed Science and Technology. 219:268-279). Thus, asfermentation progresses, dCO₂ becomes the main species, which may leadto pH decline. High levels of CO₂ reduce the buffering capability of therumen.

There are many different microbial pathways that utilize carbon dioxide.Three different bacterial carbon fixation pathways exist in nature: (1)the Calvin cycle, (2) the Wood-Ljungdahl pathway, and the reductive TCA.

High partial pressures of CO₂ in the rumen favors carbon dioxide fixersand the utilization of these pathways.

Although previously deemed negligible, modeling suggests that dCO₂concentrations can be very high, and a better predictor of ruminal pH.FIG. 7 depicts the theoretical concentrations of dissolved CO₂ withrespect to rumen pH. FIG. 8 depicts the theorized pH of the rumen whentaking into account both VFA concentrations and dCO₂ concentrations andVFA concentrations alone. The inclusion of dCO₂ in the pH prediction ismore accurate than utilizing VFAs alone. Modeling and extrapolation ofexisting data shows that as pH declines, dCO₂ concentration values couldincrease above the theoretical maximum of 50 mM. Morgante et al. (2009.Comparative Clinical Pathology. 18(3):229-232) support these models andextrapolations.

The high CO₂ concentrations in the rumen create systemic physiologicalhealth problems for the animal. (See FIG. 9 ). The ruminal microbialmetabolism can shift due to changes in the stoichiometry for VFAconcentrations that can result in lactate production, and furtherincrease histamine and LPS production. Nutritional diseases related tohigh dCO₂ concentrations cause issues in the rumen due to foaming andhigh viscosity. High dCO₂ concentrations result in rumen acidosis,increased bloat, and abomasal dysplasia. Nutritional diseases related torumen dCO₂ and high dCO₂ diffusion include respiratory and metabolicacidosis, modulated immune responses, laminitis, ketosis, liver disease,and liver abscesses. Fat and cholesterol can also mobilize to reducedCO₂ absorption, creating other deleterious physiological healthproblems for the animal. FIG. 17 indicates that many CO₂ relatedmicrobes become abundant throughout the survey experiment. ManyClostridia, Prevotella (Bacteroidales), and other acetogens utilize CO₂and hydrogen to produce acetate.

Example VI. Acidosis Challenge

Sixteen heifers are cannulated, eight controls and eight experimental.The experimental group is to receive six types of rumen microbesdirectly administered to the rumen daily.

The treatments are run as a 2×2 cross-over design with two 28 dayperiods. There are 4-6 weeks for adaptation/step up. A 28 day acidosischallenge uses elevates step-up concentrations in the diet. There is afourteen day covariate between the periods.

The ruminal pH is measured daily using an eCow eBolus. The rumen contentis sampled. 10 mL of venous blood is sampled for oximetry. The weight,contractions of the rumen, and intakes are sampled. The rumen samplesare assayed for microbial analysis, VFA analysis, and dCO₂.

The diet is as follows:

Item, % of DM Control Diet Acidosis Challenge Dry-rolled corn 66 74Dried distiller's grains 20 20 Corn silage 10 2 Premix 4 4

The heifers that received microbes daily are expected to tolerate theacidosis challenge more adeptly than heifers not administered themicrobes. The heifers that received the microbes are expected to exhibit(1) a higher ruminal and/or systemic pH during the challenge, (2) lowerconcentrations of ruminal carbon dioxide, (3) increased feed intake(more consistent intake and greater number of pounds of feed eaten, (4)greater average daily weight gain, and (5) greater final weight; ascompared to heifers not having been administered the microbes.

Example VII. Effects of Native Rumen Microorganism Supplementation onthe Ability of Heifers to Tolerate High-Grain Diets

Experimental Design: Sixteen heifers were cannulated and blocked intotwo different groups: 8 control animals, and 8 experimental animals. Theexperimental group received live cells of six different rumen bacterialstrains: Ascusbbf_24302, Ascusbbf_4, Ascusbbf_14146, Ascusbbf_154,Ascusbbf_1085, and Ascusbbf_876. Fresh cultures of each strain wereprepared, and whole cells suspended in saline were directly administeredto the rumen via cannula daily at a dose of 1E9 cells/strain/dose.Control animals received an equivalent volume of of saline daily viacannula.

The ruminal pH was measured daily using an eCow eBolus. Animal weightwas measured weekly and feed intakes were measured daily. Rumen contentwas sampled weekly to determine concentrations of VFAs and carbondioxide in the rumen, and to determine colonization of administeredstrains. Venous blood was sampled for oximetry.

Animals were stepped up to the final ration diet over 4 weeks, using 4intermediate step-up diets that gradually replaced corn silage withdry-rolled corn. The first two weeks (first two step up diets) were usedto create a baseline for blocking the animals. After these two weeks,the animals were assigned into either the experimental or control group.Microbe administration began on day 14 and continued until day 35 (21days of microbe administration).

The diet is as follows:

Item, % of DM Final ration Dry-rolled corn 66 Dried distiller's grains20 Corn silage 10 Premix 4

The diet also included a small amount of premix to add micronutrients,Rumensin, and Tylosin.

Results: Administration of microbes to heifers had a clear impact on theperformance of the animal. Animals that received microbes exhibitedhigher final weights (FIG. 25 ), and higher rumen pH (FIG. 26 ) by day35 of the experiment. Experimental animals also showed lowerconcentrations of rumen CO₂ (FIG. 227 ) as compared to control animals.As can be seen from FIG. 28 , although rumen CO₂ concentrations droppedin both groups, CO₂ concentrations in the experimental group droppedmore rapidly than the control group.

Example VIII. Carbon Fixation Pathways

The whole genomes of ten microbial strains were sequenced, annotated,and analyzed for carbon fixation-related genes.

The reductive citric acid cycle is used by some bacteria to producecarbon compounds from carbon dioxide and water. The cycle contains threecritical enzymes in the pathway: 2-oxoglutarate/2-oxoacid ferredoxinoxidoreductase (EC 1.2.7.3), ATP citrate ligase (EC 2.3.3.8), andfumarate reductase (EC 1.3.4.5, 1.3.5.1, 1.3.1.6, and 1.3.4.1 in KEGGcarbon fixation pathways). Ascusbbf_1010 and Ascusbbf_14146 contain EC1.3.5.1. Furthermore, Ascusbbf_14146 and Ascusbbf_154 contain EC1.3.5.4.

The Wood-Ljungdahl pathway, also known as the reductive acetyl-coenzymeA pathway enables some bacteria to utilize hydrogen as an electron donorand carbon dioxide as an electron acceptor and as a building block forbiosynthesis. The pathway contains three critical enzymes:carbon-monoxide dehydrogenase (EC 1.2.7.4), acetyl-CoA synthase (EC2.3.1.169), and formate dehydrogenase (EC 1.71.1.10 KEGG and 1.2.1.43PATRIC). Ascusbbf_1085 and Ascusbbf_876 both contain 1.2.7.4 and2.3.1.169. See FIG. 30 .

The Hydroxypropionate-hydroxybutyrate cycle contains three criticalenzymes: succinyl-CoA reductase (EC 1.2.1.76), acetyl-CoAC-acetyltransferase (2.3.1.9), and MalCoA lyase (4.1.3.25 KEGG and4.1.3.24 PATRIC). The ten sequenced genomes did not contain 1.2.1.76,4.1.3.25 KEGG, or 4.1.3.24 PATRIC, so it is unlikely that the strainsare utilizing this pathway. See FIG. 31 .

Several genomes suggested that the strains may be fixing carbon dioxidein conjunction with a carbohydrate, such as glucose. The strains may befixing carbon during the conversion of: acetyl-CoA+CO₂+2 reducedferredoxin+2H⁺->pyruvate+CoA=2 oxidized ferredoxin.

Example IX. Mode of Action—Highly Fermentable Diet Tolerance inRuminants

The rumen is a specialized stomach dedicated to the digestion of feedcomponents in ruminants. A diverse microbial population inhabits therumen, where their primary function revolves around converting thefibrous and non-fibrous carbohydrate components into useable sources ofenergy and protein (FIG. 3 ). In typical commercial ruminant diets,cellulose and other plant-related structural carbohydrates (e.g.hemicellulose, pectin, lignin, etc.) are one component of the feedration. This portion of the ration is considered indigestible bymammals' native metabolism, and must be degraded by microorganismsinhabiting the rumen. The cellulolytic microbes in the rumen leverageextensive enzymatic activity in order break these molecules down intosimple sugars and volatile fatty acids. This enzymatic activity iscritical to the extraction of energy from feed, and more efficientdegradation ultimately provides more energy to the animal. The solublesugars found in the non-fibrous portion of the feed are also fermentedinto gases and volatile fatty acids such as butyrate, propionate, andacetate. Volatile fatty acids arising from the digestion of both thefibrous and non-fibrous components of feed are ultimately the mainsource of energy of the ruminant.

In a feedlot setting, ruminants receive a diet with much higher levelsof concentrate and readily digestible soluble sugars to increase weightgain of the animal more rapidly. Because of the highly fermentablenature of the feed, the physiology of the rumen microbial metabolismshifts dramatically (as compared to the rumen microbial metabolism of ananimal on a more balanced diet), creating many health issues for thehost animal. High levels of fermentation will increase the build-up offermentation by-products. These by-products include various acids (e.g.lactic acid, volatile fatty acids, succinic acid, citric acid, etc.),alcohols (e.g. ethanol, propanol, etc.) and gases (e.g. hydrogen, carbondioxide, methane, hydrogen sulfide, etc.).

Accumulation of these by-products in the rumen can lead to: acidosis,bloat, panting, abomasal dysplasia, reduced buffering capacity in therumen, leaky gut, increased permeability of the gastrointestinal lining,increased histamine and LPS production, respiratory acidosis, metabolicacidosis, laminitis, ketosis, liver disease, and liver abscesses.Microorganisms strains used as products may be utilizing a variety ofmetabolic and physiological processes to maintain a more healthy rumenstate in the host animal. These microbes ultimately counter theincreased fermentation and accompanying physiological side effects. Thefunction of microorganisms in the animals that are most productive/mosttolerant of a highly fermentable diet in a feedlot setting encourage oneor more of the following:

Reduction or sequestration of carbon dioxide due to excess fermentationwithin the rumen. Microorganisms leverage a variety of differentpathways to utilize bicarbonate produced by the animal and carbondioxide produced from fermentation within the rumen. Carbon fixationpathways include the Calvin cycle, Wood-Ljundahl Pathway, Reductive TCAcycle, and variations thereof.

Reduction of the methanogen population within the rumen.Hydrogenotrophic methanogens convert CO₂ and hydrogen to methane, whichis subsequently lost to the environment due to cattle flatulence.Microbial strains that compete with methanogens and convert CO₂ into aform usable by the animal (e.g. acetate) maximize the amount of energyassimilated by the animal.

Increase production of volatile fatty acids. Volatile fatty acids arethe primary energy source for ruminants. More efficient animals tend ofhave higher concentrations of these acids in their rumen. Althoughoverall acid production increases due to the increased amount offermentation, biasing acid production towards energetically useful acidsfor the animal promotes productivity.

Increase de novo synthesis/provide vitamin B, K, and other relevantvitamins as a prebiotic. Due to the severe composition shift of therumen microbial community, vitamins typically synthesized by themicrobial community may be insufficient for both animal and microbialnutritional requirements.

Reduction of alpha diversity in the rumen microbiome. More efficientanimals have lower numbers of microbial species in their rumen microbialpopulation.

Addition of the appropriate microbial strains as a product canameliorate the effects of rapid rumen microbial fermentation andby-product accumulation, ultimately enhancing the productivity andhealth of the ruminant. Potential product microorganisms were assayed invitro for their ability to degrade various carbohydrates, and toidentify the acids produced from the degradation of these carbohydrates.(See Example XII and corresponding table). Genomic information was alsomined to identify the strains' metabolic potential to fix CO₂ andsynthesize various vitamins.

Example X. Comparative Analysis of MIC Scores from Published Work ofOther Groups

Utilizing Ascus Biosciences' technology, the performance of currentlyavailable microbial feed additive products was predicted.

Direct-fed microbial products that claim to enhance cattle performanceand prevent acidosis are available on the market. A few of theseproducts contain microorganism strains that are native rumenmicroorganisms (Megasphaera elsdenii), or are within 97% sequencesimilarity of native rumen microorganisms. Here, we've identified thespecies that are used in these products, and determined their platformscore with respect to common performance parameters: average daily gain,weight gain, feed intake, and feed efficiency (FIG. 29 ). As can be seenfrom the curve, all but one of the currently available strains fallbelow the threshold (˜0.4) used to define “useful” and “non-useful”strains. The one strain above the cutoff, Megasphaera elsdenii, hasshown a positive effect in a few studies. Other common strains used indirect fed microbial products, such as Lactobacillus animalis andPropionibacterium freudenreichii, were not similar to any native rumenmicroorganisms. Thus, scores could not be generated for thesemicroorganisms.

Lactobacillus casei: MIC 0.04587

No change in performance during finishing. Impact of a mixed culture ofLactobacillus casei and L. lactis on in vitro ruminal fermentation andthe growth of feedlot steers fed barley-based diets. Baah et al. 2009.

Megasphaera elsdenii: MIC 0.54494

Small increase in hot carcass weight. M. elsdenii on the performance ofsteers adapting to a high-concentrate diet, using three or fivetransition diets. Drouillard et al. 2012.

Higher rumen pH after carbohydrate challenge. The effects of dosingfeedlot cattle with M. elsdenii strain NCIMB 41125 prior to theintroduction of a grain-rich diet. McDaniel et al. 2007.

No effect, slight shift of acetate led to propionate production. RumenpH and fermentation characteristics in dairy cows supplemented with M.elsdenii NCIMB 41125 in early lactation. Aikman et al. 2011.

Higher feed intakes and improved ADG. Effect of ruminal administrationof the lactate-utilizing strain M. elsdenii MCIMB 41125 on abrupt orgradual transition from forage to concentrate diets. Henning et al.2010.

Higher carcass weights when used with 30 kg/ton roughage diet. Effectsof virginiamycin and monensin administered alone or together with M.elsdenii strain NCIMB 41125 on in vitro production of lactate and VFAand the effects of monensin and M. elsdenii strain NCIMB 41125 on healthand performance of feedlot steers. Leeuw et al. 2015.

Lactobacillus acidophilus: MIC 0.04839

No change in performance, but may decrease E. coli levels. Prevalence ofEscherichia coli 0157:H7 and Performance by Beef Feedlot Cattle GivenLactobacillus Direct-Fed Microbials. Brashears et al. 2003.

Slightly improved overall gain—127 kg to 130 kg (2.3%). Performance andcarcass characteristics of commercial feedlot cattle from a study ofvaccine and direct-fed microbial effects on Escherichia coli O157:H7fecal shedding. Cull et al. 2015.

No effect on performance. Direct-fed microbials containinglactate-producing bacteria influence ruminal fermentation but notlactate utilization in steers fed a high-concentrate diet. Kenney et al.2015.

No effect. Effect of Direct-Fed Microbial Dosage on the FecalConcentrations of Enterohemorrhagic Escherichia coli in Feedlot Cattle.Luedtke et al. 2016.

No effect on performance. Effect of Lactobacillus acidophilus StrainNP51 on Escherichia coli O157:H7 Fecal Shedding and FinishingPerformance in Beef Feedlot Cattle. Peterson et al. 2007.

No effect. Evaluation of Lactobacillus Fermentation Cultures in CalfFeeding Systems. Higginbotham et al. 1992.

Pediococcus acidilactici: MIC 0.14609

No effect on performance. Direct-fed microbials containinglactate-producing bacteria influence ruminal fermentation but notlactate utilization in steers fed a high-concentrate diet. Kenney et al.2015.

Enterococcus faecium: MIC 0.37215

No effect on acidosis/tolerance of high grain diet. Effects of bacterialdirect-fed microbials and yeast on site and extent of digestion, bloodchemistry, and subclinical ruminal acidosis in feedlot cattle.Beauchemin et al. 2003.

No effect on ruminal pH, blood pH, or DMI. Effects of bacterialdirect-fed microbials on ruminal fermentation, blood variables, and themicrobial populations of feedlot cattle. Ghorbani et al. 2002.

No effect on performance. Direct-fed microbials containinglactate-producing bacteria influence ruminal fermentation but notlactate utilization in steers fed a high-concentrate diet. Kenney et al.2015.

Lactobacillus reuteri: MIC 0.06366 May help reduce E. coli, no mentionof performance improvements. Lactobacillus reuteri suppresses E. coliO157:H7 in bovine ruminal fluid: Toward a pre-slaughter strategy toimprove food safety. Bertin et al. 2017.

Propionibacterium freudenreichii: MIC not Detected

Slightly improved overall gain—127 kg to 130 kg (2.3%). Performance andcarcass characteristics of commercial feedlot cattle from a study ofvaccine and direct-fed microbial effects on Escherichia coli O157:H7fecal shedding. Cull et al. 2015.

No effect on performance or carcass characteristics. Effects of liveculture of Lactobacillus acidophilus (strains NP45 and NP51) andPropionibacterium freudenreichii on performance, carcass, and intestinalcharacteristics, and Escherichia coli strain 0157 shedding of finishingbeef steers. Elam et al. 2013.

No effect on ruminal pH, blood pH, or DMI. Effects of bacterialdirect-fed microbials on ruminal fermentation, blood variables, and themicrobial populations of feedlot cattle. Ghorbani et al. 2002.

No effect on performance. Direct-fed microbials containinglactate-producing bacteria influence ruminal fermentation but notlactate utilization in steers fed a high-concentrate diet. Kenney et al.2015.

No effect.: Effect of Direct-Fed Microbial Dosage on the FecalConcentrations of Enterohemorrhagic Escherichia coli in Feedlot Cattle.Luedtke et al. 2016.

No effect on performance. Effects of increasing dose of live cultures ofLactobacillus acidophilus (Strain NP 51) combined with a single dose ofPropionibacterium freudenreichii (Strain NP 24) on performance andcarcass characteristics of finishing beef steers. Vasconcelos et al.2008.

Lactobacillus Animals: MIC not Detected

No effect on performance or carcass characteristics. Effects of liveculture of Lactobacillus acidophilus (strains NP45 and NP51) andPropionibacterium freudenreichii on performance, carcass, and intestinalcharacteristics, and Escherichia coli strain 0157 shedding of finishingbeef steers. Elam et al. 2003. L. acidophilus NP51 has been reclassifiedas Lactobacillus animalis.

No effect on performance. Effects of increasing dose of live cultures ofLactobacillus acidophilus (Strain NP 51) combined with a single dose ofPropionibacterium freudenreichii (Strain NP 24) on performance andcarcass characteristics of finishing beef steers. Vasconcelos et al.2008.

Lactobacillus plantarum: MIC not Detected

No effect on performance. Direct-fed microbials containinglactate-producing bacteria influence ruminal fermentation but notlactate utilization in steers fed a high-concentrate diet. Kenney et al.2015.

Bacillus subtilis: MIC not Detected

No effect. Evaluation of a Direct-Fed Microbial Product Effect on thePrevalence and Load of Escherichia coli O157:H7 in Feedlot Cattle.Arthur et al. 2010.

Example XI. Beef Enrichment Media Compositions

Kreb's Yeast Lactate Medium

Component g/L Yeast Extract 10.0 NaNO₃ 1.7 Sodium Lactate 46.7 KH₂PO₄ 1Na₂HPO₄ 2.4 Resazurin 1 mL Agar/Gelrite/Gellan Gum 15 DI H2O 1 L

Place the above components into a flask 2× larger than the volume beingprepared. pH to 6.8-7.0. Autoclave at 121° C. for 15 minutes, liquidcycle. Place in 47° C. for 30 minutes

MRS Medium (Originally from BD Difco Lactobacilli MR)

Component g/1000 mL Proteose Peptone No. 3 10.0 Beef Extract 10.0 YeastExtract 5.0 Glucose 20.0 Tween 80 1.0 mL Sodium Acetate 5.0 Ammoniumcitrate 2.0 Magnesium Sulfate 0.1 Manganese Sulfate 0.05 Agar 15.0L-Cysteine HCl 0.5 0.1% Rezasurin 1.0 mL DI H2O 1000 mL

Place the aforementioned components into a flask 2× larger than thevolume being prepared. Adjust pH to 6.8 with HCl or NaOH. Autoclave at121° C. for 15 minutes, liquid cycle.

M2 Salts

Component g/L (NH₄)₂SO₄ 0.674 MgSO₄ 7H₂O 0.035 K₂HPO₄ 0.348 KH₂PO₄ 0.3470.1% Resazurin 1 mL DI H₂O Up to 1 L

Place the aforementioned components into a flask 2× larger than thevolume being prepared. Adjust pH to 6.8. Place in 47° C. for 30 minutes.Add L-cysteine hydrochloride (100 mM) to liquid media. Sterile filterany additives from the following list to create amended medias.

Component Molarity (mM) g/L Fructose 13.9 2.5 Arabinose 16.7 2.5Cellulose 7.3 2.5 Xylose 79.9 12.0 Maltose 2.86 0.978 Galactose 13.882.5 Cellobiose 10 3.42 Ground Corn** — 0.5 Sodium Lactate (60% Soln)233.8 20 mLs Amino Acid Soln 1 — 100 mLs Fatty Acid Soln 2 — 20 mLsWolfes Mineral Solution — 1.0 mL **Add to media before autoclaving

Amino Acid Solution 1 (50 mL)

Component Molarity (mM) g/50 mL Glutamine 100.0 .731 Glycine 100.0 .375Proline 100.0 .576 DI H₂O — Up to 50 mL

Fatty Acid Solution 2 (50 mL)

Component 1.1 - Molarity (mM) uL/50 mL Propionic Acid 9.40 35.0Isovaleric Acid 0.906 5.0 Methylbutyric ACid 0.916 5.0 DI H₂O — Up to 50mL

Wolfe's Mineral Solution

Component g/L MgSO₄ 7H₂O 3.0 Nitrilotriacetic acid 1.5 NaCl 1.0 MnSO₄H₂O 0.5 CaCl₂ 0.1 CoCl₂ 6H₂O 0.1 FeSO₄ 7H₂O 0.1 ZnSO₄ 7H₂O 0.1 AlK(SO₄)₂12H₂O 0.01 CuSO₄ 5H₂O 0.01 H₃BO₃ 0.01 NaMoO₄ 2H₂O 0.01 DI H2O To 1 L

MBM

Component g/L 100x Wolfes Minerals 1.0 mL Ammonium Sulfate  0.90Cellobiose 4.0 Sodium Bicarbonate 7.5 Sodium Carbonate 4.0 0.1%Resazurin 1 mL VFA Soln 48 mL DI H2O 1 L

Place the aforementioned components into a flask 2× the size of thevolume being made.

To create modified MBM (MBM mod) media, add the following components:

Component g/L KH₂PO₄ 1.0 K₂HPO₄ 1.0

pH to 6.8-7.0. Autoclave at 121° C. for 15 minutes, liquid cycle. Placein 47° C. water bath for 30 minutes. Sterile filter any of the followingadditives to create amended media:

Component Molarity (mM) g/L Xylan 30.0  4.98 Fructose 13.88 2.5 Cellulose  7.30 2.5 

VFA Solution

Component g/L Acetic Acid 40 mL Isobutyric Acid  2 mL Isovaleric Acid  2mL Valeric Acid  2 mL 2-Methylbutyric acid  2 mL

RCM

BD Difco Dehydrated Culture Media: Reinforced Clostridial Medium

Component g/L Peptone 10 Beef Extract 10 Yeast Extract 3 Dextrose 5Sodium Chloride 5 Soluble Starch 1 Cysteine HCl 0.5 Sodium Acetate 3Agar 0.5

Place the aforementioned components into a flask 2× larger than thevolume being made. pH to 6.8-7.0. Autoclave at 121° C. for 15 minutes,liquid cycle.

Trypic Soy Broth (TSB)

Add the following components into a flask 2× larger than the volumebeing made. Add 500 mL of deionized water. Add stir bar to flask andplace on stir plate. Stir while adding 30 g/L of Tryptic Soy Broth(Sigma Aldrich T8907). Top off to 850 mL with deionized water. Add 1 mLof 0.01% resazurin indicator. Autoclave at 121° C. for 15 minutes,liquid cycle. Place flask in hot bath at 47° C. for 30 minutes.

TSBHK

Sterile filter (0.2 micrometer) 10 mL hemin (0.05%) and 0.2 mL vitaminK1 solution.

To create stock solutions:

Hemin Solution (100 mLs) Component grams/volumes Hemin (Sigma AldrichH9039) 50 mg 1M NaOH 1.0 mLs dI H2O  99 mLs

Vitamin K1 Solution (30 mLs) Component mLs Vitamin K1 (Sigma AldrichV3501) 0.15 95% EtOH 30

TSB+5% Sheep's Blood

Add 50 mL of defibrinated sheep's blood to 950 mL of TSB.

VL55/BC (Modified DSMZ 1266)

Place the following components into a flask 2× larger than the volumebeing made.

Component mL/L 2- 1.95 g Morpholinoethanesulfonic Acid (MES) 20 mM MgSO₄10.0 30 mM CaCl₂ 10.0 20 mM (NH₄)₂HPO₄ 10.0 Selenite-Tungstate Soln 1.0Trace Element Soln SL10 1.0 0.1% Resazurin 1.0 Di H₂O 960

Once all components are in solution, aliquot 50 mL into separate 120 mLserum vials. Each vial is bubbled under N₂ gas for 3 minutes at 15 psi.All vials are stoppered with grey stops and crimped with aluminum tearoff crimps. Head space of every vial is sparged, needle in/out, for 2minutes under N₂ gas. All vials are labeled and autoclaved at 121° C.for 20 minutes under fast cycle. When vials have finished autoclavingand cooled, the following three sets of components are added at thefollowing concentration to every vial, via syringe, syringe filter 0.2micrometers, and needle.

Component mL/L 100× Wolfe's Minerals 10.0 1000× Wolfe's Vitamins 1.0 BCSolution 40.0 5% Na₂S 10.0 NaOH 10.0 Na₂SeO × 5H₂O 1.0 Na₂WO₄ × 2H₂O10.0 Di H₂O 1000 Component mg/L HCl (25%; 7.7M) 10.0 mLs FeCl₂ × 4H₂O1.50 g ZnCl₂ 70.0 MnCl₂ × 4H₂O 100.0 H₃BO₃ 6.0 CoCl₂ × 6H₂O 190.0 CuCl₂× 2H₂O 2.0 NiCl₂ × 6H₂O 24.0 Na₂MoO₄ × 2H₂O 36.0 Di H₂O 990.0

First dissolved FeCl₂ in the HCL, then dilute in water, add and dissolvethe other salts. Finally make up to volume.

Component g/50 mLs Bile Salts 1.0 g Creatine .282

Rumen Winogradsky Column

Clarified Rumen Fluid Creation

-   -   (a) Obtain approximately 2 L of unfiltered rumen fluid.    -   (b) Aliquot into 250 mL conicals.    -   (c) Spin at 4300 RPM for 30 minutes at 4° C.        -   (i) Remove the supernatant (lx Clarified Rumen Fluid) in an            anaerobic chamber with 5:20:75, H2:CO₂:N₂ gas. Store half            the 1× Clarified Rumen Fluid in an airtight container at            4° C. until ready to use.        -   (ii) Remove the pellet (Rumen solids) from all conicals and            store in an airtight container at 4° C. until ready to use.    -   (d) Aliquot the 1× Clarified Rumen Fluid into 50 mL conicals.    -   (e) Spin 1× Clarified Rumen Fluid at 13200 RPM for 30 minutes        -   (i) Remove supernatant (2× Clarified Rumen Fluid) in an            anaerobic chamber and store in an airtight container at            4° C. until ready to use.        -   (ii) Discard the pellet.

Creation of Column

-   -   (a) Bring sterile Winogradsky Column, fresh beef Total Mixed        Ration (TMR), Beef Rumen Sample, Rumen Solids, lx Clarified        Rumen Fluid and 2× Clarified Rumen Fluid into an anaerobic        chamber.    -   (b) Tightly pack approximately 100-mLs of Rumen Solids into the        bottom of the column.    -   (c) Pour approximately 150-mLs 1× Clarified Rumen Fluid on top        of the Rumen Solids.    -   (d) Pour Approximately 150-mLs 2× Clarified Rumen Fluid on top        of the 1× Rumen Fluid.    -   (e) Seed column with approximately 10-mLs raw beef rumen sample.    -   (f) Add 50-mLs TMR to the top of the column.    -   (g) Incubate at 39° C. in a well ventilated incubator.

Basal Salts Medium with Sodium Sulfate (BSMS)

Prepare the following solutions and filter sterilize and then store at4° C.

Solution I

Component g/L Na₂SO₄ 5.0 NaCl 2.0 (NH₄)₂SO₄ 3.0 KH₂PO₄ 3.0 CaCl₂ 2H₂O0.6 MgSO₄ 7H₂O 0.6

Solution II

K₂HPO₄ 3.0

Media Preparation

Component g/L Solution I 150 mL Solution II 150 mL Yeast extract 1.50Sodium Bicarbonate 6.0 

Bring up to 1 L. Adjust pH to 7.0+/−0.05. Autoclave for 15 minutes at121° C. under slow exhaust. After autoclave, add sterile L-cysteine (100micromolar).

RAMM Salts with Vitamins, Minerals, and Sodium Sulfide

Component g/500 mL KH₂PO₄ 0.11 K₂HPO₄ 0.08 NH₄Cl 0.265 NaHCO₃ 0.6 DIH₂O500 mL

After autoclaving, add 5 mL of 100× Wolfe's vitamin mix. 5 mL of 5%sodium sulfide.

Example XII. Analysis of Rumen Microbes for Volatile Fatty AcidProduction and Carbon Source Use

A. Carbon Source Use

To assess the ability of the strains to metabolize various carbonsources, OD600 was used to measure the growth of strains over time inboth minimal and rich media. Minimal media conditions demonstrate whatcompounds a strain can use as its sole carbon source, while the richmedia iteration shows which carbon sources a strain can use under ideal,rumen-like conditions.

A single colony from each of the desired strains (on anaerobic agarplates) was inoculated into 1 ml of anaerobic RAMM salts. This wasvortexed briefly and 10 μL was inoculated into a rich and minimal media,spiked with a chosen carbon source.

Strains were inoculated anaerobically into 1 mL of media in the 2 mlwell of a 96 well plate. Carbon sources included arabinose, fructose,dextrose, galactose, xylose, cellobiose and starch. Carbon sources forthe minimal media conditions were made at a 20 g/L concentration in RAMMsalts, and filter sterilized through a 0.22 μm polyethersulfonemembrane. Starch was prepared at a concentration of 2 g/L andautoclaved. Carbon sources for the rich media conditions were made inRAMM salts at a 200 g/L concentration and 100 μl of sterile carbonsource was added to 900 μl of rich media.

Plates were incubated in the dark anaerobically at 37° C. 100 μLaliquots of each well were periodically read at 600 nm over a two-weekperiod on a “Synergy H4 hybrid plate reader”. Strain ID was confirmedprior to inoculation with Illumina sequencing. Duplicate entriesrepresent strains within 97% of the listed strain ID.

Minimal strain id Arabinose Fructose Starch Dextrose Galactose XyloseCellobiose Ascusbbf_24302 + N/A − + + + + Ascusbbf_24302 + N/A − + + + +Ascusbbf_10712 − N/A − − − − − Ascusbbf_4 − N/A − − − − − Ascusbbf_4 −N/A − − − − − Ascusbbf_951 − N/A − − − − − Ascusbbf_951 − − N/A − − − −Ascusbbf_951 − N/A − − − − − Ascusbbf_14146 + N/A − + + + +Ascusbbf_14146 − N/A − + − − − Ascusbbf_14146 − − N/A − − − −Ascusbbf_154 − N/A − − − − − Ascusbbf_1010 + N/A + + + + − Ascusbbf_154− N/A − − − − − Ascusbbf_24302 + N/A + + + + + Ascusbbf_24302 +N/A + + + + + Ascusbbf_1085 − N/A − − − − − Ascusbbf_14146 − N/A + + + +− Ascusbbf_951 − N/A − − − − − Ascusbbf_951 − N/A − − − − − Ascusbbf_951− N/A − − − − − Ascusbbf_1085 − N/A − − − − − Ascusbbf_876 − N/A − − − −− Ascusbbf_10712 − N/A − − − − − Ascusbbf_10712 − N/A − − − − −Ascusbbf_10109 − − N/A − − − −

Rich strain id Arabinose Fructose Dextrose Galactose Xylose CellobioseAscusbbf_24302 + + + + + + Ascusbbf_24302 + + + + + + Ascusbbf_10712− + + − − − Ascusbbf_4 N/A N/A N/A N/A N/A N/A Ascusbbf_4 − − − + − −Ascusbbf_951 N/A N/A N/A N/A N/A N/A Ascusbbf_951 + + + + − +Ascusbbf_951 − + + − − − Ascusbbf_14146 N/A N/A N/A N/A N/A N/AAscusbbf_14146 N/A N/A N/A N/A N/A N/A Ascusbbf_14146 − + − − − −Ascusbbf_154 N/A N/A N/A N/A N/A N/A Ascusbbf_1010 + + + + − −Ascusbbf_154 + + + + + + Ascusbbf_24302 + + + + + +Ascusbbf_24302 + + + + + + Ascusbbf_1085 − + − + − − Ascusbbf_14146 N/AN/A N/A N/A N/A N/A Ascusbbf_951 − − − − − − Ascusbbf_951 − − − − − −Ascusbbf_951 − − − − − − Ascusbbf_1085 − + + + − + Ascusbbf_876 − − − −− − Ascusbbf_10712 − + + − − − Ascusbbf_10712 − + + − − −Ascusbbf_10109 + − − − − −

B. Volatile Fatty Acid (VFA) Production

To assess the ability of the strains or enrichments to produce volatilefatty acids, HPLC was used to measure the concentrations of acetate,butyrate, and propionate in spent media.

For pure isolates, a single colony from each of the desired strains (onsolid anaerobic media) was inoculated into the strain's preferred richmedia. Enrichments were inoculated from fresh rumen sample into adesired media.

Cultures and medium blanks were incubated at their optimal conditionsuntil significant growth was visible in the cultures. Absorbance readswere taken at 600 and 420 nm to determine the growth of each culture.

Pure culture strain IDs were confirmed with Illumina sequencing.Enrichments and their corresponding rumen sample inocula were Illuminasequenced to determine the presence or absence of target strains. Thesesequencing datasets were integrated with cell count data to determine iftarget strains grew in vitro.

An aliquot of each culture was sterile filtered through 0.22 micrometerpolyethersulfone membrane into a sterile acid washed 15 mL glass samplevial to be analyzed by HPLC. HPLC reactions were performed at MichiganState University Bioeconomy Institute. Concentrations of acetate,butyrate, and propionate were quantified for the cultures and mediablanks. HPLC parameters: The column is BioRad Aminex HPX-87H, 60° C.,0.5 mL/min mobile phase 0.00325 N H2504, 500 psi, 35C RI detector, 45min run time, injection volume 5 μL.

Strain ID pyruvic glucose succinic lactic glycerol acetic propionicbutyric ethanol 1-Butanol Ascusbbf_1007A 0 0 0 0 0 + + + − 0Ascusbbf_100A 0 − + − + + + 0 0 0 Ascusbbf_100A 0 0 0 0 + − 0 0 + 0Ascusbbf_100A 0 − + − − + − + − 0 Ascusbbf_100A 0 0 0 + 0 + 0 0 0 0Ascusbbf_100B 0 0 0 0 − − 0 + 0 0 Ascusbbf_100B 0 − + 0 0 + + + + 0Ascusbbf_100C 0 − + − + + + 0 0 0 Ascusbbf_100C 0 0 0 0 − − 0 + 0 0Ascusbbf_100C 0 0 0 0 + + 0 + + 0 Ascusbbf_100C 0 − + 0 0 + + 0 + 0Ascusbbf_100C 0 0 0 0 + − 0 0 + 0 Ascusbbf_100C 0 0 0 0 0 + 0 0 0 0Ascusbbf_100C 0 0 0 + 0 + 0 0 0 0 Ascusbbf_100D 0 − + − + + + 0 0 0Ascusbbf_100D 0 0 0 0 − − 0 + 0 0 Ascusbbf_100D 0 0 0 0 0 + + + − 0Ascusbbf_100D 0 − + − − + − + − 0 Ascusbbf_100E 0 − + − + + + 0 0 0Ascusbbf_100E 0 0 0 0 − − 0 + 0 0 Ascusbbf_100E 0 0 0 0 + + 0 + + 0Ascusbbf_100E 0 − + 0 0 + + 0 + 0 Ascusbbf_100E 0 0 0 0 + − 0 0 + 0Ascusbbf_100E 0 0 0 0 0 + 0 0 0 0 Ascusbbf_100F 0 0 0 0 0 + + + − 0Ascusbbf_100G 0 − + 0 0 + + 0 + 0 Ascusbbf_10109A + − + − 0 + + + − 0Ascusbbf_10109B + − + − 0 + + + − 0 Ascusbbf_10109C 0 − + − + + + 0 0 0Ascusbbf_10109C 0 0 0 0 + − 0 0 + 0 Ascusbbf_10109D 0 0 0 0 + − 0 0 + 0Ascusbbf_10109E 0 0 0 0 + − 0 0 + 0 Ascusbbf_10109E 0 − + − − + − + − 0Ascusbbf_10109F 0 0 0 0 + − 0 0 + 0 Ascusbbf_10109G 0 0 0 0 + − 0 0 + 0Ascusbbf_10109G 0 − + + − + + + − 0 Ascusbbf_10109H 0 0 0 0 + − 0 0 + 0Ascusbbf_10109H 0 − + − − + − + − 0 Ascusbbf_10109I 0 − + − − + − + − 0Ascusbbf_1010A + − + − 0 + + + − 0 Ascusbbf_1010A + − 0 + − + + 0 + 0Ascusbbf_1010B 0 − + − + + + 0 0 0 Ascusbbf_1010B 0 0 0 0 − − 0 + 0 0Ascusbbf_1010B 0 0 0 0 + + 0 + + 0 Ascusbbf_1010B 0 − + 0 0 + + + + 0Ascusbbf_1010B 0 0 0 0 + − 0 0 + 0 Ascusbbf_1010B 0 0 0 0 0 + 0 0 0 0Ascusbbf_1010B 0 0 + + + + + 0 + 0 Ascusbbf_1010B 0 0 0 + 0 + 0 0 + 0Ascusbbf_1010C 0 0 0 0 − − 0 + 0 0 Ascusbbf_1010C 0 0 0 0 + + 0 + + 0Ascusbbf_1010C 0 − + 0 0 + + + + 0 Ascusbbf_1010C 0 0 0 0 + 0 0 + 0Ascusbbf_1010C 0 0 0 0 0 + 0 0 0 0 Ascusbbf_1010C 0 0 + + + + + 0 + 0Ascusbbf_1010C 0 0 0 + 0 + 0 0 0 0 Ascusbbf_1010D 0 − + − + + + 0 0 0Ascusbbf_1010D 0 0 0 0 − − 0 + 0 0 Ascusbbf_1010D 0 0 0 0 + + 0 + + 0Ascusbbf_1010D 0 − + 0 0 + + + + 0 Ascusbbf_1010D 0 0 0 0 + − 0 0 + 0Ascusbbf_1010D 0 − + + + + 0 0 + 0 Ascusbbf_1010D 0 0 + + + + + 0 + 0Ascusbbf_1010D 0 0 0 + 0 + 0 0 0 0 Ascusbbf_1010E 0 − + − + + + 0 0 0Ascusbbf_1010E 0 0 0 0 − − 0 + 0 0 Ascusbbf_1010E 0 0 0 0 0 + + + − 0Ascusbbf_1010E 0 − + − + + + + + 0 Ascusbbf_1010E 0 0 0 + 0 + 0 0 0 0Ascusbbf_1010F 0 − + − + + + 0 0 0 Ascusbbf_1010F 0 0 0 0 − − 0 + 0 0Ascusbbf_1010F 0 0 0 0 0 + + + − 0 Ascusbbf_1010F 0 0 0 + 0 + 0 0 0 0Ascusbbf_1010G 0 − + − + + + 0 0 0 Ascusbbf_1010G 0 0 0 0 − − 0 + 0 0Ascusbbf_1010G 0 0 0 0 + + 0 + + 0 Ascusbbf_1010G 0 − + 0 0 + + + + 0Ascusbbf_1010G 0 0 0 0 + − 0 0 + 0 Ascusbbf_1010G 0 − + + + + 0 0 + 0Ascusbbf_1010G 0 0 + + + + + 0 + 0 Ascusbbf_1010G 0 0 0 + 0 + 0 0 0 0Ascusbbf_1010H 0 − + − + + + 0 0 0 Ascusbbf_1010H 0 0 0 0 0 + + + − 0Ascusbbf_1010I 0 − + − + + + 0 0 0 Ascusbbf_1010I 0 0 0 0 0 + + + − 0Ascusbbf_1010I 0 0 + + + + + 0 + 0 Ascusbbf_1010J 0 0 0 0 + + 0 + + 0Ascusbbf_1010J 0 0 0 0 0 + 0 0 0 0 Ascusbbf_1010J 0 0 0 + 0 + 0 0 0 0Ascusbbf_1034A 0 0 0 + 0 + 0 0 + 0 Ascusbbf_1034A 0 0 0 + 0 + 0 0 + 0Ascusbbf_1034B 0 0 0 + 0 + 0 0 + 0 Ascusbbf_1034B 0 0 0 + 0 + 0 0 + 0Ascusbbf_104A 0 0 0 − 0 + 0 0 + − Ascusbbf_104B 0 0 0 0 − − 0 + 0 0Ascusbbf_104B 0 − + 0 0 + + + + 0 Ascusbbf_104B 0 0 0 0 + − 0 0 + 0Ascusbbf_104B 0 − + − − + − + − 0 Ascusbbf_104C 0 0 0 0 − − 0 + 0 0Ascusbbf_104C 0 − + 0 0 + + + + 0 Ascusbbf_104C 0 0 0 0 + − 0 0 + 0Ascusbbf_104C 0 − + − − + − + − 0 Ascusbbf_104D 0 0 0 0 − − 0 + 0 0Ascusbbf_104E 0 0 0 0 − − 0 + 0 0 Ascusbbf_104E 0 0 0 0 0 + + + − 0Ascusbbf_104E 0 − + 0 0 + + + + 0 Ascusbbf_104F 0 − + − + + + 0 0 0Ascusbbf_104F 0 − + 0 0 + + + + 0 Ascusbbf_104G 0 − + − − + − + − 0Ascusbbf_104H 0 − + − + + + + + 0 Ascusbbf_104I 0 − + − + + + + + 0Ascusbbf_10576A 0 0 0 0 + − 0 0 + 0 Ascusbbf_10576A 0 − + − − + − + − 0Ascusbbf_10576A 0 0 + + + + + 0 + 0 Ascusbbf_10576B 0 0 0 0 + − 0 0 + 0Ascusbbf_10576B 0 − + − − + − + − 0 Ascusbbf_10576B 0 0 + + + + + 0 + 0Ascusbbf_10576B 0 0 0 + 0 + 0 0 0 0 Ascusbbf_10576C 0 0 0 0 + − 0 0 + 0Ascusbbf_10576C 0 − + − − + − + − 0 Ascusbbf_10576C 0 0 + + + + + 0 + 0Ascusbbf_10576D 0 0 0 0 + − 0 0 + 0 Ascusbbf_10576E 0 0 + + + + + 0 + 0Ascusbbf_106863A 0 0 0 + 0 + 0 0 + 0 Ascusbbf_106863A 0 0 0 + 0 + 0 0 +0 Ascusbbf_106863B 0 0 0 + 0 + 0 0 + 0 Ascusbbf_106863B 0 0 0 + 0 + 00 + 0 Ascusbbf_10712A 0 − + − + + + 0 0 0 Ascusbbf_10712A 0 − + 00 + + + + 0 Ascusbbf_10712A 0 − + + + + 0 0 + 0 Ascusbbf_10712A + − + −0 + + + − 0 Ascusbbf_10712B + − + − 0 + + + − 0 Ascusbbf_10712C 0 − +− + + + 0 0 0 Ascusbbf_10712C 0 − + 0 0 + + 0 + 0 Ascusbbf_10712C 0 0 00 + − 0 0 + 0 Ascusbbf_10712D 0 − + − + + + 0 0 0 Ascusbbf_10712D 0 − +0 0 + + + + 0 Ascusbbf_10712E 0 − + 0 0 + + + + 0 Ascusbbf_10712E 0 0 00 + − 0 0 + 0 Ascusbbf_10712F 0 − + − + + + 0 0 0 Ascusbbf_10712F 0 − 0− − 0 0 + + 0 Ascusbbf_10712F 0 − + 0 0 + + + + 0 Ascusbbf_10712F 0 − +− − + − + − 0 Ascusbbf_10712F 0 − 0 − − 0 0 + + 0 Ascusbbf_10712G 0 − +− + + + 0 0 0 Ascusbbf_10712G 0 − + 0 0 + + + + 0 Ascusbbf_10712G 0 0 00 + − 0 0 + 0 Ascusbbf_10712G 0 − + − − + − + − 0 Ascusbbf_10712G 0 − 0− − 0 0 + + 0 Ascusbbf_10712H 0 − + − + + + 0 0 0 Ascusbbf_10712H 0 − +0 0 + + + + 0 Ascusbbf_10712H 0 0 0 0 + − 0 0 + 0 Ascusbbf_10712H 0 − +− − + − + − 0 Ascusbbf_10712H 0 0 0 + 0 + 0 0 0 0 Ascusbbf_10712I 0 − +0 0 + + + + 0 Ascusbbf_10712I 0 − + + + + 0 0 + 0 Ascusbbf_1085A 0 − +− + + + 0 0 0 Ascusbbf_1085A 0 0 0 0 0 + + + − 0 Ascusbbf_1085A 0 0 00 + − 0 0 + 0 Ascusbbf_1085A 0 0 0 0 0 + 0 0 0 0 Ascusbbf_1085A + − + −0 + + + − 0 Ascusbbf_1085A + − 0 + − + 0 + − 0 Ascusbbf_1085B 0 − +− + + + 0 0 0 Ascusbbf_1085B 0 − + − + + + + + 0 Ascusbbf_1085B 0 0 0 00 + 0 0 0 0 Ascusbbf_1085B 0 0 + + + + + 0 + 0 Ascusbbf_1085B + − 0 +0 + 0 + + 0 Ascusbbf_1085C 0 − + − + + + 0 0 0 Ascusbbf_1085C 0 0 0 0 −− 0 + 0 0 Ascusbbf_1085C 0 0 0 0 0 + + + − 0 Ascusbbf_1085C 0 − + 00 + + 0 + 0 Ascusbbf_1085C 0 0 0 0 + − 0 0 + 0 Ascusbbf_1085C 0 0 0 00 + 0 0 0 0 Ascusbbf_1085C 0 0 0 + 0 + 0 0 + 0 Ascusbbf_1085C 0 0 0 +0 + 0 0 + 0 Ascusbbf_1085D 0 0 0 0 − − 0 + 0 0 Ascusbbf_1085D 0 0 0 00 + + + − 0 Ascusbbf_1085D 0 − + 0 0 + + + + 0 Ascusbbf_1085D 0 0 0 0 +− 0 0 + 0 Ascusbbf_1085D 0 0 0 0 0 + 0 0 0 0 Ascusbbf_1085D 0 0 0 + 0 +0 0 0 0 Ascusbbf_1085E 0 − + − + + + 0 0 0 Ascusbbf_1085E 0 0 0 0 − −0 + 0 0 Ascusbbf_1085E 0 0 0 0 0 + + + − 0 Ascusbbf_1085E 0 − + 00 + + + + 0 Ascusbbf_1085E 0 0 0 0 + − 0 0 + 0 Ascusbbf_1085E 0 0 0 00 + 0 0 0 0 Ascusbbf_1085E 0 0 0 + 0 + 0 0 0 0 Ascusbbf_1085E + − 0 +0 + 0 + + 0 Ascusbbf_1085F 0 − + − + + + 0 0 0 Ascusbbf_1085F 0 0 0 0 +− 0 0 + 0 Ascusbbf_1085F 0 0 0 + 0 + 0 0 + 0 Ascusbbf_1085F 0 0 0 + 0 +0 0 + 0 Ascusbbf_1085G 0 − + − + + + 0 0 0 Ascusbbf_1085G 0 − + − − +− + − 0 Ascusbbf_1085H 0 − + − + + + 0 0 0 Ascusbbf_1085H 0 − + − − +− + − 0 Ascusbbf_1085I 0 − + − + + + 0 0 0 Ascusbbf_1085I 0 − + − − +− + − 0 Ascusbbf_1085J 0 − + − + + + 0 0 0 Ascusbbf_1085J 0 − +− + + + + + 0 Ascusbbf_1085K 0 − + − + + + + + 0 Ascusbbf_1103A 0 − +− + + + 0 0 0 Ascusbbf_1103A 0 0 0 0 + + 0 + + 0 Ascusbbf_1103B 0 0 0 00 + + + − 0 Ascusbbf_1103B 0 0 + + + + + 0 + 0 Ascusbbf_1103B 0 0 0 +0 + 0 0 + 0 Ascusbbf_1103C 0 0 0 0 0 + + + − 0 Ascusbbf_1103C 0 0 0 +0 + 0 0 + 0 Ascusbbf_1103C 0 0 0 + 0 + 0 0 + 0 Ascusbbf_1103D 0 0 00 + + 0 + + 0 Ascusbbf_1103D 0 − + 0 0 + + 0 + 0 Ascusbbf_1103D 0 0 0 +0 + 0 0 + 0 Ascusbbf_1103D 0 0 0 + 0 + 0 0 + 0 Ascusbbf_1103E 0 0 00 + + 0 + + 0 Ascusbbf_1103E 0 0 + + + + + 0 + 0 Ascusbbf_1103E 0 0 0 +0 + 0 0 + 0 Ascusbbf_1103F 0 0 0 + 0 + 0 0 + 0 Ascusbbf_1103G 0 0 0 +0 + 0 0 + 0 Ascusbbf_1103G 0 0 0 + 0 + 0 0 + 0 Ascusbbf_1103H 0 0 0 +0 + 0 0 + 0 Ascusbbf_1103H 0 0 0 + 0 + 0 0 + 0 Ascusbbf_1103I 00 + + + + + 0 + 0 Ascusbbf_1103I 0 0 0 + 0 + 0 0 + 0 Ascusbbf_113152A 0− + − − + − + − 0 Ascusbbf_1136A 0 0 0 0 − − 0 + 0 0 Ascusbbf_1136A 0 00 0 + + 0 + + 0 Ascusbbf_1136A 0 − + 0 0 + + + + 0 Ascusbbf_1136A 0 0 00 + − 0 0 + 0 Ascusbbf_1136A 0 − + − − + − + − 0 Ascusbbf_1136B 0 0 0 0− − 0 + 0 0 Ascusbbf_1136B 0 0 0 0 0 + + + − 0 Ascusbbf_1136B 0 − + 00 + + 0 + 0 Ascusbbf_1136B 0 0 0 0 + − 0 0 + 0 Ascusbbf_1136B 0 − + −− + − + − 0 Ascusbbf_1136B 0 0 0 + 0 + 0 0 0 0 Ascusbbf_1136C 0 − +− + + + 0 0 0 Ascusbbf_1136C 0 0 0 0 − − 0 + 0 0 Ascusbbf_1136C 0 0 0 00 + + + − 0 Ascusbbf_1136C 0 − + 0 0 + + + + 0 Ascusbbf_1136C 0 0 0 0 +− 0 0 + 0 Ascusbbf_1136C 0 − + − − + − + − 0 Ascusbbf_1136D 0 0 0 0 − −0 + 0 0 Ascusbbf_1136D 0 0 0 0 + + 0 + + 0 Ascusbbf_1136D 0 − + 00 + + + + 0 Ascusbbf_1136D 0 0 0 0 + − 0 0 + 0 Ascusbbf_1136D 0 0 0 00 + 0 0 0 0 Ascusbbf_1136D 0 0 0 + 0 + 0 0 0 0 Ascusbbf_1136E 0 0 0 0 −− 0 + 0 0 Ascusbbf_1136E 0 0 0 0 + + 0 + + 0 Ascusbbf_1136E 0 0 0 0 + −0 0 + 0 Ascusbbf_1136E 0 − + − − + − + − 0 Ascusbbf_1136F 0 − + 00 + + + + 0 Ascusbbf_11823A 0 0 0 0 0 + + + − 0 Ascusbbf_11823B 0 0 0 00 + + + − 0 Ascusbbf_11823B 0 − + − + + + + + 0 Ascusbbf_11823B 0 0 00 + − 0 0 + 0 Ascusbbf_11823C 0 − + − − + − + − 0 Ascusbbf_118A 0 0 00 + − 0 0 + 0 Ascusbbf_118A 0 0 0 + 0 + 0 0 + 0 Ascusbbf_118A 0 0 0 +0 + 0 0 + 0 Ascusbbf_118B 0 0 0 + 0 + 0 0 + 0 Ascusbbf_118B 0 0 0 + 0 +0 0 + 0 Ascusbbf_118C 0 0 0 + 0 + 0 0 + 0 Ascusbbf_118C 0 0 0 + 0 + 00 + 0 Ascusbbf_1207A 0 − + − + + + 0 0 0 Ascusbbf_1207A 0 − + 0 0 + +0 + 0 Ascusbbf_1207A 0 0 0 0 + − 0 0 + 0 Ascusbbf_1207A 0 − + − − + − +− 0 Ascusbbf_1207B 0 − + − + + + 0 0 0 Ascusbbf_1207B 0 − + − + + + + +0 Ascusbbf_1207C 0 − + − + + + 0 0 0 Ascusbbf_1207D 0 0 0 0 − − 0 + 0 0Ascusbbf_1207D 0 0 0 0 0 + + + − 0 Ascusbbf_1207E 0 0 0 0 − − 0 + 0 0Ascusbbf_1207E 0 0 0 0 0 + + + − 0 Ascusbbf_1207F 0 0 0 0 − − 0 + 0 0Ascusbbf_1207F 0 0 0 0 0 + + + − 0 Ascusbbf_1207F 0 − + 0 0 + + 0 + 0Ascusbbf_1207G 0 − + − + + + 0 0 0 Ascusbbf_1207G 0 0 0 0 − − 0 + 0 0Ascusbbf_1207G 0 0 0 0 + + 0 + + 0 Ascusbbf_1207G 0 − + 0 0 + + 0 + 0Ascusbbf_1207G 0 0 0 0 + − 0 0 + 0 Ascusbbf_1207G 0 0 0 0 0 + 0 0 0 0Ascusbbf_1207G 0 0 0 + 0 + 0 0 0 0 Ascusbbf_1207H 0 − + − + + + 0 0 0Ascusbbf_1207H 0 0 0 0 − − 0 + 0 0 Ascusbbf_1207I 0 − + − + + + 0 0 0Ascusbbf_1207I 0 0 0 0 − − 0 + 0 0 Ascusbbf_1207I 0 0 0 0 + + 0 + + 0Ascusbbf_1207I 0 0 0 0 + − 0 0 + 0 Ascusbbf_1207I 0 0 0 0 0 + 0 0 0 0Ascusbbf_1207J 0 − + − + + + 0 0 0 Ascusbbf_1207J 0 − + 0 0 + + 0 + 0Ascusbbf_1207J 0 0 0 0 + − 0 0 + 0 Ascusbbf_1207K 0 − + − + + + 0 0 0Ascusbbf_1207K 0 0 0 0 + − 0 0 + 0 Ascusbbf_1207K 0 − + − − + − + − 0Ascusbbf_1207L 0 0 0 0 + − 0 0 + 0 Ascusbbf_120A 0 0 + + + + + 0 + 0Ascusbbf_120B 0 0 + + + + + 0 + 0 Ascusbbf_120C 0 0 + + + + + 0 + 0Ascusbbf_121971A 0 0 0 0 0 + + + − 0 Ascusbbf_1238A 0 − + − + + + 0 0 0Ascusbbf_1238A 0 0 0 0 − − 0 + 0 0 Ascusbbf_1238A 0 0 0 0 + + 0 + + 0Ascusbbf_1238A 0 0 0 0 0 + 0 0 0 0 Ascusbbf_1238A 0 0 + + + + + 0 + 0Ascusbbf_1238A 0 0 0 + 0 + 0 0 0 0 Ascusbbf_1238B 0 0 0 0 − − 0 + 0 0Ascusbbf_1238B 0 0 0 0 + + 0 + + 0 Ascusbbf_1238B 0 0 0 0 0 + 0 0 0 0Ascusbbf_1238B 0 0 0 + 0 + 0 0 0 0 Ascusbbf_1238C 0 − + − + + + 0 0 0Ascusbbf_1238C 0 0 0 0 − − 0 + 0 0 Ascusbbf_1238C 0 0 0 0 + + 0 + + 0Ascusbbf_1238C 0 − + 0 0 + + + + 0 Ascusbbf_1238C 0 0 0 0 0 + 0 0 0 0Ascusbbf_1238C 0 0 + + + + + 0 + 0 Ascusbbf_1238C 0 0 0 + 0 + 0 0 0 0Ascusbbf_1238D 0 − + − + + + 0 0 0 Ascusbbf_1238D 0 0 + + + + + 0 + 0Ascusbbf_1238D 0 0 0 + 0 + 0 0 0 0 Ascusbbf_1273A 0 0 0 0 + − 0 0 + 0Ascusbbf_1273A 0 0 + + + + + 0 + 0 Ascusbbf_1273A 0 0 0 + 0 + 0 0 + 0Ascusbbf_1273B 0 0 0 + 0 + 0 0 + 0 Ascusbbf_1273C 0 0 + + + + + 0 + 0Ascusbbf_1273C 0 0 0 + 0 + 0 0 + 0 Ascusbbf_1273D 0 0 + + + + + 0 + 0Ascusbbf_1273D 0 0 0 + 0 + 0 0 + 0 Ascusbbf_130A 0 0 0 0 + − 0 0 + 0Ascusbbf_130A 0 − + − − + − + − 0 Ascusbbf_130A 0 0 0 + 0 + 0 0 + 0Ascusbbf_130A 0 0 0 + 0 + 0 0 + 0 Ascusbbf_130B 0 0 0 0 + − 0 0 + 0Ascusbbf_130C 0 0 0 0 + − 0 0 + 0 Ascusbbf_130C 0 − + − − + − + − 0Ascusbbf_130D 0 0 0 0 + − 0 0 + 0 Ascusbbf_130D 0 − + − − + − + − 0Ascusbbf_130E 0 0 0 0 + − 0 0 + 0 Ascusbbf_130F 0 0 0 0 + − 0 0 + 0Ascusbbf_130F 0 − + − − + − + − 0 Ascusbbf_130G 0 0 + + + + + 0 + 0Ascusbbf_1325058A 0 0 0 0 0 + + + − 0 Ascusbbf_1325058A 0 0 0 0 + − 00 + 0 Ascusbbf_1325058A 0 0 0 + 0 + 0 0 + 0 Ascusbbf_1325058A 0 0 0 +0 + 0 0 + 0 Ascusbbf_1325058B 0 0 0 0 + + 0 + + 0 Ascusbbf_1325058B 0− + 0 0 + + 0 + 0 Ascusbbf_1325058B 0 0 0 0 + − 0 0 + 0Ascusbbf_1325058B 0 − + − − + − + − 0 Ascusbbf_1325058B 0 0 + + + + +0 + 0 Ascusbbf_1325058C 0 0 0 0 + − 0 0 + 0 Ascusbbf_1325058D 0 0 0 0 +− 0 0 + 0 Ascusbbf_1325058E 0 − + − − + − + − 0 Ascusbbf_1325058F 00 + + + + + 0 + 0 Ascusbbf_13543A 0 − + − + + + 0 0 0 Ascusbbf_13543B 00 0 0 + + 0 + + 0 Ascusbbf_13543B 0 0 0 0 0 + 0 0 0 0 Ascusbbf_13543C 00 0 0 + + 0 + + 0 Ascusbbf_13543C 0 − + 0 0 + + 0 + 0 Ascusbbf_13543C 00 0 0 0 + 0 0 0 0 Ascusbbf_13543C 0 0 0 + 0 + 0 0 + 0 Ascusbbf_13543C 00 0 + 0 + 0 0 + 0 Ascusbbf_13543D 0 0 0 0 + + 0 + + 0 Ascusbbf_13543D 00 0 0 0 + 0 0 0 0 Ascusbbf_13543E 0 − + − + + + 0 0 0 Ascusbbf_13543E 00 0 0 + + 0 + + 0 Ascusbbf_13543E 0 − + 0 0 + + 0 + 0 Ascusbbf_13543E 00 0 + 0 + 0 0 + 0 Ascusbbf_13543E 0 0 0 + 0 + 0 0 + 0 Ascusbbf_13717A 00 + + + + + 0 + 0 Ascusbbf_13717A 0 0 0 + 0 + 0 0 + 0 Ascusbbf_13717B 00 + + + + + 0 + 0 Ascusbbf_13717B 0 0 0 + 0 + 0 0 + 0 Ascusbbf_13717C 00 + + + + + 0 + 0 Ascusbbf_13717C 0 0 0 + 0 + 0 0 + 0 Ascusbbf_1372985A0 0 0 0 0 + + + − 0 Ascusbbf_1372985A + − + − 0 + + + − 0Ascusbbf_1372985B 0 − + − + + + 0 0 0 Ascusbbf_1372985B 0 0 0 0 0 + + +− 0 Ascusbbf_1372985B 0 − + 0 0 + + 0 + 0 Ascusbbf_1372985C 0 − +− + + + 0 0 0 Ascusbbf_1372985C 0 0 0 0 0 + + + − 0 Ascusbbf_1372985D 00 0 0 − − 0 + 0 0 Ascusbbf_1372985D 0 0 0 0 + + 0 + + 0Ascusbbf_1372985D 0 − + 0 0 + + + + 0 Ascusbbf_1372985D 0 0 0 0 + 0 0 +0 Ascusbbf_1372985D 0 0 0 0 0 + 0 0 0 0 Ascusbbf_1372985E 0 0 0 0 − −0 + 0 0 Ascusbbf_1372985E 0 0 0 0 + + 0 + + 0 Ascusbbf_1372985E 0 0 00 + − 0 0 + 0 Ascusbbf_1372985E 0 0 0 0 0 + 0 0 0 0 Ascusbbf_1372985F 00 0 0 0 + + + − 0 Ascusbbf_1372985I 0 − + − + + + 0 0 0Ascusbbf_1372985I 0 0 0 0 0 + + + − 0 Ascusbbf_1372985L 0 0 0 0 0 + + +− 0 Ascusbbf_1372985M 0 0 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0Ascusbbf_92A 0 − + − − + − + − 0 Ascusbbf_92B 0 0 0 0 − − 0 + 0 0Ascusbbf_92B 0 0 0 0 + + 0 + + 0 Ascusbbf_92B 0 0 0 0 + − 0 0 + 0Ascusbbf_92B 0 0 0 0 0 + 0 0 0 0 Ascusbbf_92C 0 0 0 0 − − 0 + 0 0Ascusbbf_92C 0 − + − − + − + − 0 Ascusbbf_92D 0 0 0 0 − − 0 + 0 0Ascusbbf_92D 0 0 0 0 + − 0 0 + 0 Ascusbbf_92D 0 0 0 0 0 + 0 0 0 0Ascusbbf_92E 0 0 0 0 − − 0 + 0 0 Ascusbbf_92F 0 0 0 0 − − 0 + 0 0Ascusbbf_92F 0 0 0 0 + − 0 0 + 0 Ascusbbf_92F 0 0 0 0 0 + 0 0 0 0Ascusbbf_92H 0 0 0 0 0 + + + − 0 Ascusbbf_92H 0 0 0 0 + − 0 0 + 0Ascusbbf_92K 0 0 0 0 0 + + + − 0 Ascusbbf_92K 0 0 + + + + + 0 + 0Ascusbbf_92K 0 0 0 + 0 + 0 0 0 0 Ascusbbf_92L 0 − + − − + − + − 0Ascusbbf_930A 0 0 + + + + + 0 + 0 Ascusbbf_930A 0 0 0 + 0 + 0 0 0 0Ascusbbf_930B 0 0 + + + + + 0 + 0 Ascusbbf_930B 0 0 0 + 0 + 0 0 + 0Ascusbbf_944A + − + − 0 + + + − 0 Ascusbbf_944B 0 0 0 0 + + 0 + + 0Ascusbbf_944B 0 0 0 0 + − 0 0 + 0 Ascusbbf_944B 0 0 + + + + + 0 + 0Ascusbbf_944B 0 0 0 + 0 + 0 0 + 0 Ascusbbf_944C 0 0 0 0 + − 0 0 + 0Ascusbbf_944C 0 0 + + + + + 0 + 0 Ascusbbf_944D 0 0 0 0 + − 0 0 + 0Ascusbbf_944D 0 − + − − + − + − 0 Ascusbbf_944E 0 0 0 0 + − 0 0 + 0Ascusbbf_944F 0 0 0 0 + − 0 0 + 0 Ascusbbf_944G 0 0 0 0 + − 0 0 + 0Ascusbbf_944G 0 0 0 + 0 + 0 0 + 0 Ascusbbf_944G 0 0 0 + 0 + 0 0 + 0Ascusbbf_951A 0 − + − + + + + + 0 Ascusbbf_951A + − + − 0 + + + − 0Ascusbbf_951A 0 − 0 + + + 0 0 + 0 Ascusbbf_951B 0 − + − + + + 0 0 0Ascusbbf_951B 0 0 0 0 − − 0 + 0 0 Ascusbbf_951B 0 0 0 0 + + 0 + + 0Ascusbbf_951B 0 − + 0 0 + + 0 + 0 Ascusbbf_951B 0 0 0 0 + − 0 0 + 0Ascusbbf_951B 0 − + + + + 0 0 + 0 Ascusbbf_951B 0 0 0 + 0 + 0 0 + 0Ascusbbf_951B 0 0 0 + 0 + 0 0 0 0 Ascusbbf_951B − − − + − + + + − 0Ascusbbf_951B 0 − 0 + 0 0 0 0 + 0 Ascusbbf_951B 0 − 0 + 0 0 0 0 + 0Ascusbbf_951C 0 − + − + + + 0 0 0 Ascusbbf_951C 0 0 0 0 − − 0 + 0 0Ascusbbf_951C 0 0 0 0 + + 0 + + 0 Ascusbbf_951C 0 − + 0 0 + + 0 + 0Ascusbbf_951C 0 0 0 0 + − 0 0 + 0 Ascusbbf_951C 0 − + + + + 0 0 + 0Ascusbbf_951C 0 0 0 + 0 + 0 0 + 0 Ascusbbf_951C 0 0 0 + 0 + 0 0 0 0Ascusbbf_951D 0 − + − + + + 0 0 0 Ascusbbf_951D 0 0 0 0 − − 0 + 0 0Ascusbbf_951D 0 0 0 0 0 + + + − 0 Ascusbbf_951D 0 − + 0 0 + + + + 0Ascusbbf_951D 0 0 0 0 + − 0 0 + 0 Ascusbbf_951E 0 − + − + + + 0 0 0Ascusbbf_951E 0 − + − + + + + + 0 Ascusbbf_951E 0 0 0 + 0 + 0 0 + 0Ascusbbf_951E 0 0 0 + 0 + 0 0 + 0 Ascusbbf_951F 0 − + − + + + 0 0 0Ascusbbf_951G 0 − + − + + + 0 0 0 Ascusbbf_95A 0 0 0 0 + − 0 0 + 0Ascusbbf_95A 0 − + − − + − + − 0 Ascusbbf_95B 0 0 0 0 + − 0 0 + 0Ascusbbf_95B 0 − + − − + − + − 0 Ascusbbf_95B 0 0 + + + + + 0 + 0Ascusbbf_95B 0 0 0 + 0 + 0 0 + 0 Ascusbbf_95C 0 0 0 0 + − 0 0 + 0Ascusbbf_95C 0 − + − − + − + − 0 Ascusbbf_95D 0 0 0 0 + − 0 0 + 0Ascusbbf_95D 0 − + − − + − + − 0 Ascusbbf_95D 0 0 + + + + + 0 + 0Ascusbbf_95D 0 0 0 + 0 + 0 0 + 0 Ascusbbf_95E 0 0 0 + 0 + 0 0 + 0Ascusbbf_95E 0 0 0 + 0 + 0 0 + 0 Ascusbbf_95F 0 0 0 + 0 + 0 0 + 0Ascusbbf_95F 0 0 0 + 0 + 0 0 + 0 Ascusbbf_95G 0 0 0 + 0 + 0 0 + 0Ascusbbf_95G 0 0 0 + 0 + 0 0 + 0 Ascusbbf_95H 0 0 0 + 0 + 0 0 + 0Ascusbbf_95H 0 0 0 + 0 + 0 0 + 0 Ascusbbf_9770A 0 0 0 0 + + 0 + + 0Ascusbbf_9770A 0 − + 0 0 + + 0 + 0 Ascusbbf_9770A 0 0 0 0 + − 0 0 + 0Ascusbbf_9770A 0 0 0 0 0 + 0 0 0 0 Ascusbbf_9770B 0 0 0 0 + + 0 + + 0Ascusbbf_9770B 0 0 0 0 + − 0 0 + 0 Ascusbbf_9770B 0 0 0 0 0 + 0 0 0 0Ascusbbf_9770C 0 0 0 0 + − 0 0 + 0 Ascusbbf_9770C 0 0 0 + 0 + 0 0 + 0Ascusbbf_9770C 0 0 0 + 0 + 0 0 + 0 Ascusbbf_9770D 0 0 0 0 + − 0 0 + 0Ascusbbf_9770D 0 − + − − + − + − 0 Ascusbbf_9770E 0 0 0 0 + − 0 0 + 0Ascusbbf_9770E 0 − + − − + − + − 0 Ascusbbf_9770F 0 0 0 0 + − 0 0 + 0Ascusbbf_9770F 0 − + − − + − + − 0 Ascusbbf_9770G 0 0 0 0 + − 0 0 + 0Ascusbbf_9770G 0 − + − − + − + − 0 Ascusbbf_9770H 0 0 0 0 + − 0 0 + 0Ascusbbf_9770H 0 − + − − + − + − 0 Ascusbbf_9770H 0 0 0 + 0 + 0 0 + 0Ascusbbf_9770H 0 0 0 + 0 + 0 0 + 0 Ascusbbf_983757A 0 0 0 + 0 + 0 0 + 0Ascusbbf_983757A 0 0 0 + 0 + 0 0 + 0 Ascusbbf_983757B 0 0 0 + 0 + 0 0 +0 Ascusbbf_983757B 0 0 0 + 0 + 0 0 + 0 Ascusbbf_983757C 0 0 0 + 0 + 00 + 0 Ascusbbf_983757C 0 0 0 + 0 + 0 0 + 0

Example XIII. Effects of Native Rumen Microorganism Supplementation onFeed Conversion Ratio of Heifers on High-Grain Diets

Experimental Design: Sixteen heifers were cannulated and blocked intotwo different groups: 8 control animals, and 8 experimental animals. Theexperimental group received live cells of six different rumen bacterialstrains: Ascusbbf_24302, Ascusbbf_4, Ascusbbf_14146, Ascusbbf_154,Ascusbbf_1085, and Ascusbbf_876. Fresh cultures of each strain wereprepared, and whole cells suspended in saline were directly administeredto the rumen via cannula daily at a dose of 1E9 cells/strain/dose.Control animals received an equivalent volume of saline daily viacannula.

The ruminal pH was measured daily using an eCow eBolus. Animal weightwas measured weekly and feed intakes were measured daily. Rumen contentwas sampled weekly to determine concentrations of VFAs and carbondioxide in the rumen, and to determine colonization of administeredstrains. Venous blood was sampled for oximetry. Feed conversion ratiowas calculated by dividing weight gain by the amount of feed consumed.

Animals were stepped up to the final ration diet over 4 weeks, using 4intermediate step-up diets that gradually replaced corn silage withdry-rolled corn. The first two weeks (first two step up diets) were usedto create a baseline for blocking the animals. After these two weeks,the animals were assigned into either the experimental or control group.Microbe administration began on day 14 and continued until day 56 (42days of microbe administration). On day 48, all heifers underwent anacidosis challenge for 8 days. The acidosis challenge was induced byincreasing the amount of corn in the diet of the animals.

The diet is as follows:

Control Acidosis Item, % of DM Diet Challenge Dry-rolled corn 66 74Dried distiller's grains 20 20 Corn silage 10  2 Premix  4  4The diet also included a small amount of premix to add micronutrients,Rumensin, and Tylosin.

Results

Administration of microbes to heifers had a clear impact on theperformance of the animal. Intakes and weights of the animals weremeasured throughout the step up and acidosis challenge. Feed conversionratio (FCR) was calculated by dividing the amount of feed consumed bythe animal over a specific amount of time by the weight gain of theanimal over the same time period. Animals that received microbesexhibited improved feed conversion during the last phase of step up andthe acidosis challenge as compared to control animals (FIG. 32 ).Animals in the control group required roughly 6 lbs of feed to create 1lb of body weight, while experimental animals required roughly 4 lbs offeed to create 1 lb of body weight.

Numbered Embodiments of the Disclosure

1. A microbial composition comprising at least one microbial strainselected from Table 1 and/or Table 2.

2. A microbial composition comprising at least one microbial strain,wherein the at least one microbial strain comprises a 16S rRNA sequenceselected from SEQ ID NOs:1-100.

3. The microbial composition of claim 2, wherein the at least onemicrobial strain comprises Ascusbbf_6176.

4. The microbial composition of claim 2, wherein the at least onemicrobial strain comprises Ascusbbf_22143.

5. The microbial composition of claim 2, wherein the at least onemicrobial strain comprises Ascusbbf_4483.

6. The microbial composition of claim 2, wherein the at least onemicrobial strain comprises Ascusbbf_13543.

7. The microbial composition of claim 2, wherein the at least onemicrobial strain comprises Ascusbbf_152.

8. The microbial composition of claim 2, wherein the at least onemicrobial strain comprises Ascusbbf_707 and Ascusbbf_1238.

9. The microbial composition of claim 2, wherein the at least onemicrobial strain comprises Ascusbbf_4691, Ascusbbf_5588, andAscusbbf_1238.

10. The microbial composition of any one of claims 1-9, wherein saidmicrobial composition is encapsulated.

11. A composition comprising:

-   -   (a) a microbial composition of any one of claims 1-10, and    -   (b) an acceptable carrier.

12. The composition of claim 11, wherein the microbial composition isencapsulated.

13. The composition of claim 11, wherein the encapsulated microbialcomposition comprises a polymer selected from a saccharide polymer, agarpolymer, agarose polymer, protein polymer, and lipid polymer.

14. The composition of claim 11, wherein the acceptable carrier isselected from the group consisting of: edible feed grade material,mineral mixture, water, glycol, molasses, and corn oil.

15. The composition of claim 11, wherein the at least two microbialstrains forming the microbial composition are present in the compositionat 102 to 1015 cells per gram of said composition.

16. The composition of claim 11, wherein said composition is (a) mixedwith animal feed, (b) mixed with animal drinking water, or (c)administered onto animal feed.

17. A method of imparting at least one improved trait upon an animal,said method comprising administering the composition of claim 11 to saidanimal.

18. The method of claim 17, wherein said animal is a ruminant.

19. The method of claim 17, wherein said steer is an Angus breed ofcattle, or a hybrid or cross thereof.

20. The method of claim 18, wherein the administration comprises (a)injecting the composition into rumen of the animal, (b) administering abolus of the composition, or (c) drenching the animal with thecomposition.

21. The method of claim 17, wherein said composition is administered atleast once per month.

22. The method of claim 21, wherein said composition is administered atleast once per week.

23. The method of claim 22, wherein said composition is administered atleast once per day.

24. The method of claim 17, wherein the administration occurs each timethe animal is fed.

25. The method of claim 17, wherein the administration is a rectaladministration.

26. The method of claim 25, wherein the rectal administration comprisesinserting a suppository comprising the composition into the rectum ofthe animal.

27. The method of claim 17, wherein the administration is an oraladministration.

28. The method of claim 27, wherein the oral administration comprisesadministering the composition in combination with the animal's feed,water, medicine, or vaccination.

29. The method of claim 27, wherein the oral administration comprisesapplying the composition in a gel or viscous solution to a body part ofthe animal, wherein the animal ingests the composition.

30. The method of claim 17, wherein the administration comprisesspraying the composition onto the animal feed, and wherein the animalingests the animal feed.

31. The method of claim 17, wherein said at least one improved trait isselected from the group consisting of: an increase in weight; anincrease of musculature; an increase of fatty acid concentration in thegastrointestinal tract; an increase of fatty acid concentration in therumen; a decrease in lactate concentration in the rumen; an improvedefficiency in feed utilization and digestibility; an improved feedefficiency; an improved average daily gain; an improved dry matterintake; an increase in polysaccharide and lignin degradation; anincrease in fat, starch, and/or protein digestion; an increase in fattyacid concentration in the rumen; pH balance in the rumen, an increase invitamin availability; an increase in mineral availability; an increasein amino acid availability; a reduction in methane and/or nitrous oxideemissions; a reduction in manure production; an improved efficiency ofnitrogen utilization; an improved efficiency of phosphorous utilization;an increased resistance to colonization of pathogenic microbes thatcolonize cattle; reduced mortality; increased production ofantimicrobials; increased clearance of pathogenic microbes; increasedresistance to colonization of pathogenic microbes that colonize cattle;increased resistance to colonization of pathogenic microbes that infecthumans; and any combination thereof; reduced incidence and/or prevalenceof acidosis or bloat; reduced body temperature; improved rumen wallintegrity; faster adaptation to a high grain diet; improved tolerance ofa high grain diet; wherein said increase or reduction is determined bycomparing against an animal not having been administered saidcomposition.

32. The method of claim 31, wherein said increase in weight is anincrease by at least 0.1%.

33. The method of claim 31, wherein said reduction in manure productionis a reduction by at least 0.1%.

34. The method of claim 31, wherein said increase in polysaccharidedegradation is an increase in the degradation of lignin, cellulose,and/or hemicellulose.

35. The method of claim 31, wherein said increase in fatty acidconcentration is an increase in acetic acid, propionic acid, and/orbutyric acid.

36. The composition of claim 11, wherein the at least one microbialstrain exhibit an increased utility that is not exhibited when said atleast one microbial strain occurs alone, or when said at least onemicrobial strain is present at naturally occurring concentrations.

37. The composition of claim 11, wherein the at least one microbialstrain exhibits a synergistic effect on imparting at least one improvedtrait in an animal.

38. A feedlot cattle feed supplement capable of increasing a desirablephenotypic trait in feedlot cattle, the feed supplement comprising:

-   -   (a) a microbial composition of any one of claims 1-9 present at        a concentration that does not occur naturally in said cattle,        and    -   (b) an acceptable carrier.

39. The feedlot cattle feed supplement of claim 38, wherein themicrobial composition is encapsulated.

40. An isolated microbial strain selected from any one of the microbialstrains in Table 1 and/or Table 2.

41. An isolated microbial strain selected from the group consisting of:

-   -   (a) Ascusbbf_6176 deposited as ATCC Accession Deposit No.        PTA-______;    -   (b) Ascusbbf_22143 deposited as ATCC Accession Deposit No.        PTA-______;    -   (c) Ascusbbf_4883 deposited as ATCC Accession Deposit No.        PTA-______;    -   (d) Ascusbbf_13543 deposited as ATCC Accession Deposit No.        PTA-______;    -   (e) Ascusbbf_152 deposited as ATCC Accession Deposit No.        PTA-______;    -   (f) Ascusbbf_707 deposited as ATCC Accession Deposit No.        PTA-______;    -   (g) Ascusbbf_1238 deposited as ATCC Accession Deposit No.        PTA-______;    -   (h) Ascusbbf_5588 deposited as ATCC Accession Deposit No.        PTA-______; and    -   (i) Ascusbbf_4691 deposited as ATCC Accession Deposit No.        PTA-______.

42. An isolated microbial strain comprising a polynucleotide sequencesharing at least 90% sequence identity with any one of SEQ ID NOs:1-100.

43. A substantially pure culture of an isolated microbial strainaccording to any one of claims 40 to 42.

44. A method of modulating the microbiome of a cow, the methodcomprising administering the composition of claim 12.

45. The method of claim 44, wherein the administration of thecomposition imparts at least one improved trait upon the steer, bull,cow, or heifer.

46. The method of claim 45, wherein the at least one improved trait isselected from the group consisting of: an increase in weight; anincrease of musculature; an increase of fatty acid concentration in thegastrointestinal tract; an increase of fatty acid concentration in therumen; a decrease in lactate concentration in the rumen; an improvedefficiency in feed utilization and digestibility; an improved feedefficiency; an improved average daily gain; an improved dry matterintake; an increase in polysaccharide and lignin degradation; anincrease in fat, starch, and/or protein digestion; an increase in fattyacid concentration in the rumen; pH balance in the rumen, an increase invitamin availability; an increase in mineral availability; an increasein amino acid availability; a reduction in methane and/or nitrous oxideemissions; a reduction in manure production; an improved efficiency ofnitrogen utilization; an improved efficiency of phosphorous utilization;an increased resistance to colonization of pathogenic microbes thatcolonize cattle; reduced mortality; increased production ofantimicrobials; increased clearance of pathogenic microbes; increasedresistance to colonization of pathogenic microbes that colonize cattle;increased resistance to colonization of pathogenic microbes that infecthumans; and any combination thereof; reduced incidence and/or prevalenceof acidosis or bloat; reduced body temperature; improved rumen wallintegrity; faster adaptation to a high grain diet; improved tolerance ofa high grain diet; wherein said increase or reduction is determined bycomparing against an animal not having been administered saidcomposition.

47. The method of claim 46, wherein said increase in weight is anincrease by at least 0.1%.

48. The method of claim 46, wherein said increase in feed efficiency isan increase by at least 0.1%.

49. The method of claim 46, wherein said increase in polysaccharidedegradation is an increase in the degradation of lignin, cellulose,and/or hemicellulose.

50. The method of claim 46, wherein said increase in fat digestion,starch digestion, and/or protein digestion is an increase by at least0.1%.

51. The method of claim 46, wherein said increase in fatty acidconcentration is an increase in acetic acid, propionic acid, and/orbutyric acid.

52. The method of claim 45, wherein the modulation of the microbiome isan increase in the proportion of the at least one microbial strain ofthe microbiome, wherein the increase is measured relative to a steer,bull, cow, or heifer that did not have the at least one microbial strainadministered.

53. The method of claim 45, wherein the modulation of the microbiome isa decrease in the proportion of the microbial strains present in themicrobiome prior to the administration of the composition, wherein thedecrease is measured relative to the microbiome of the steer, bull, cow,or heifer prior to the administration of the composition.

54. A method of increasing resistance of cattle to the colonization ofpathogenic microbes, the method comprising the administration of thecomposition of claim 11, wherein the pathogen is unable to colonize thegastrointestinal tract of the steer, bull, cow, or heifer.

55. The method of treating cattle for the presence of at least onepathogenic microbe, the method comprising the administration of thecomposition of claim 11.

56. The method of claim 55, wherein after administration of thecomposition the relative abundance of the at least one pathogenicmicrobe decreases to less than 5% relative abundance in thegastrointestinal tract.

57. The method of claim 56, wherein the relative abundance of the atleast one pathogenic microbe decreases to least than 0.1% relativeabundance in the gastrointestinal tract.

58. The method of claim 56, wherein the at least one pathogenic microbeis undetectable in the gastrointestinal tract.

59. The method of claim 58, wherein less than 10 days postadministration of the composition the at least one pathogenic microbe isundetectable in the gastrointestinal tract.

60. The method of claim 58, wherein within 5-15 days post administrationof the composition the at least one pathogenic microbe is undetectablein the gastrointestinal tract.

61. The method of any one of claims 54-61, wherein the at least onepathogenic microbe is selected from: Clostridium perfringens,Clostridium botulinum, Salmonella typi, Salmonella typhimurium,Salmonella enterica, Salmonella pullorum, Erysipelothrix insidiosa,Campylobacter jejuni, Campylobacter coli, Campylobacter lari, Listeriamonocytogenes, Streptococcus agalactiae, Streptococcus dysgalactiae,Corynebacterium bovis, Mycoplasma sp., Citrobacter sp., Enterobactersp., Pseudomonas aeruginosa, Pasteurella sp., Bacillus cereus, Bacilluslicheniformis, Streptococcus uberis, Staphylococcus aureus, andpathogenic strains of enteropathogenic, enteroinvasive, orenterohemorrhagic Escherichia coli, and Staphylococcus aureus.

62. The method of claim 61, wherein the at least one pathogenic microbeis selected from enteropathogenic E. coli, enteroinvasive E. coli, orenterohemorrhagic E. coli.

64. The method of claim 17, wherein said composition is administered tothe animal in a high grain diet adaptation phase.

65. The method of claim 17, wherein said composition is administered tothe animal on a feed lot diet.

67. The method of claim 17, wherein said composition is administered tothe animal on a grain intensive diet.

In aspects, the aforementioned microbial species—that is, a purifiedmicrobial population that comprises a bacteria with a 16S nucleic acidsequence, and/or a fungi with an ITS nucleic acid sequence, which is atleast about 97% identical to a nucleic acid sequence selected from thegroup consisting of: SEQ ID NOs: 1-5,368—are members of a Markush group,as the present disclosure illustrates that the members belong to a classof microbes characterized by various physical and functional attributes,which can include any of the following: a) the ability to convert acarbon source into a volatile fatty acid such as acetate, butyrate,propionate, or combinations thereof; b) the ability to degrade a solubleor insoluble carbon source; c) the ability to impart a decreased methaneoutput in feedlot cattle administered the microbe(s); d) the ability tomodulate the microbiome of the gastrointestinal tract of feedlot cattleadministered the microbe; e) the ability to be formulated into ashelf-stable composition; f) the ability to exhibit a decrease in feedconversion ratio and/or increase the weight gain, and/or increase theaverage daily gain in feedlot cattle having been administered themicrobe(s); g) the ability to impart a decrease in pathogen-associatedlesion formation in the gastrointestinal tract; h) the ability to imparta decrease in pathogenic microbes in the gastrointestinal tract; i)possessing a MIC score of at least about 0.4 if a bacteria. j) theability to reduce acidosis and/or bloat in feedlot cattle administeredthe microbe; k) the ability to reduce carbon dioxide concentrations inthe rumen of feedlot cattle administered the microbe; l) the ability toincrease pH and/or improve the buffering capability of the rumen offeedlot cattle administered the microbe; and/or m) reduce lactateconcentrations in the rumen of feedlot cattle administered the microbe.Thus, the members of the Markush group possess at least one property incommon, which can be responsible for their function in the claimedrelationship.

As used herein “shelf-stable” refers to a functional attribute and newutility acquired by the microbes formulated according to the disclosure,which enable said microbes to exist in a useful/active state outside oftheir natural environment in the gastrointestinal tract (i.e. a markedlydifferent characteristic). Thus, shelf-stable is a functional attributecreated by the formulations/compositions of the disclosure and denotingthat the microbe formulated into a shelf-stable composition can existoutside the gastrointestinal tract and under ambient conditions for aperiod of time that can be determined depending upon the particularformulation utilized, but in general means that the microbes can beformulated to exist in a composition that is stable under ambientconditions for at least a few days and generally at least one week.Accordingly, a “shelf-stable feedlot cattle supplement” is a compositioncomprising one or more microbes of the disclosure, said microbesformulated in a composition, such that the composition is stable underambient conditions for at least one week, meaning that the microbescomprised in the composition (e.g. whole cell, spore, or lysed cell) areable to impart one or more beneficial phenotypic properties to feedlotcattle when administered (e.g. increased weight gain, improvedgastrointestinal health, and/or modulation of the gastrointestinalmicrobiome).

In some embodiments, the isolated microbial strains of the presentdisclosure further encompass mutants thereof. In some embodiments, thepresent disclosure further contemplates microbial strains having all ofthe identifying characteristics of the presently disclosed microbialstrains.

TABLE 11 Budapest Treaty Deposits of the Disclosure Depository AccessionNumber Date of Deposit NRRL B-67550 Feb. 7, 2018 NRRL B-67551 Feb. 7,2018 NRRL B-67552 Feb. 7, 2018 NRRL B-67553 Feb. 7, 2018 NRRL B-67554Feb. 7, 2018 NRRL B-67555 Feb. 7, 2018 ATCC PTA-12942  Feb. 14, 2018ATCC PTA-125033 Mar. 22, 2018 ATCC PTA-125040 Mar. 29, 2018 ATCCPTA-125041 Mar. 29, 2018 ATCC PTA-125042 Mar. 29, 2018 ATCC PTA-125049Apr. 4, 2018 ATCC PTA-125050 Apr. 4, 2018 ATCC PTA-125051 Apr. 5, 2018ATCC PTA-125052 Apr. 5, 2018

INCORPORATION BY REFERENCE

All references, articles, publications, patents, patent publications,and patent applications cited herein are incorporated by reference intheir entireties for all purposes.

However, mention of any reference, article, publication, patent, patentpublication, and patent application cited herein is not, and should notbe taken as, an acknowledgment or any form of suggestion that theyconstitute valid prior art or form part of the common general knowledgein any country in the world.

Ascus Biosciences, Inc. is the assignee/applicant of the followingpatents and/or patent publications that relate generally to the subjectmatter of microbial compositions, microbial ensembles, methods of makingthe compositions and ensembles, and methods of administering such: PCTPublication Nos. WO2016210251A1, WO2017120495A1, and WO2017181203A1;U.S. Pat. Nos. 9,540,676 and 9,938,558; and U.S. Pregrant PublicationNos. US20170342457A1, US20160376627A1, US20170107557A1, US20170196922A1,and US20170196921A1.

1. A method for improving one or more desirable traits in a ruminant,the method comprising administering to the ruminant an effective amountof a microbial composition comprising: (a) a purified population of oneor more bacteria comprising a 16S nucleic acid sequence sharing at least97% sequence identity to SEQ ID NO: 17, SEQ ID NO: 75, and/or SEQ ID NO:86; and (b) a carrier suitable for ruminant administration.
 2. Themethod of claim 1, further comprising one or more bacteria sharing atleast 97% sequence identity to a 16S nucleic acid sequence selectedfrom: SEQ ID NOs: 1-16, 18-74, 76-85, 87-5993.
 3. The method of claim 1,further comprising one or more bacteria sharing at least 97% sequenceidentity to a 16S nucleic acid sequence selected from: SEQ ID NOs: 1-16,18-74, 76-85, 87-136, 5369-5378, 5398, 5425, 5450, 5460, 5462, 5473,5474, 5483, 5502, 5511, 5517, 5519, 5526, 5576, 5582, 5589, 5598, 5614,5619, 5621, 5627, 5629, 5631, 5663, 5670, 5726, 5746, 5757, 5764, 5777,5797, 5802, 5868, 5872, 5930, 5947, 5949, 5955, 5956, 5971, 5973, 5977,5989, and
 5991. 4. The method of claim 1, wherein the one or morebacteria comprises a 16S nucleic acid sequence of SEQ ID NO: 17, SEQ IDNO: 75, and/or SEQ ID NO:
 86. 5. The method of claim 1, wherein the oneor more bacteria comprises a 16S nucleic acid sequence of SEQ ID NO: 17,SEQ ID NO: 75, and SEQ ID NO:
 86. 6. The method of claim 1, wherein theone or more bacteria comprises a 16S nucleic acid sequence of SEQ ID NO:5457, SEQ ID NO: 5644, and/or SEQ ID NO:
 5709. 7. The method of claim 1,wherein the one or more bacteria comprises a 16S nucleic acid sequenceof SEQ ID NO: 5457, SEQ ID NO: 5644, and SEQ ID NO:
 5709. 8. The methodof claim 1, wherein the one or more bacteria comprises a 16S nucleicacid sequence of SEQ ID NO: 5457 and SEQ ID NO:
 5644. 9. The method ofclaim 1, wherein the one or more bacteria comprises a 16S nucleic acidsequence of SEQ ID NO: 5457 and SEQ ID NO:
 5709. 10. The method of claim1, wherein the one or more bacteria comprises a 16S nucleic acidsequence of SEQ ID NO: 5644 and SEQ ID NO:
 5709. 11. The method of claim1, wherein the one or more bacteria comprises a 16S nucleic acidsequence of SEQ ID NO:
 5457. 12. The method of claim 1, wherein the oneor more bacteria comprises a 16S nucleic acid sequence of SEQ ID NO:5644.
 13. The method of claim 1, wherein the one or more bacteriacomprises a 16S nucleic acid sequence of SEQ ID NO:
 5709. 14. The methodof claim 1, wherein the microbial composition comprises a purifiedpopulation of one or more bacteria deposited as B-67553, B-67550, and/orB-67552.
 15. The method of claim 1, wherein the microbial composition isa tablet, a capsule, a pill, a feed additive, a powder, a foodingredient, a food preparation, a food supplement, a water additive, athermostable-additive, a moisture-resistant additive, a pre-pelletedfeed additive, a pelleted feed additive, a post-pelleting-applied feedadditive, a consumable solution, a consumable spray additive, aconsumable solid, a consumable gel, an injection, a suppository, adrench, a bolus, or combinations thereof.
 16. The method of claim 1,wherein the one or more bacteria are encapsulated.
 17. The method ofclaim 16, wherein the one or more bacteria are encapsulated in one ormore of a polymer, carbohydrate, sugar, sugar alcohol, surfactant,plastic, glass, polysaccharide, lipid, wax, oil, fatty acid, amino acid,or glyceride.
 18. The method of claim 1, wherein the one or morebacteria comprises at least 10² cells.
 19. The method of claim 1,wherein the one or more bacteria are in the form of spores.
 20. Themethod of claim 1, wherein the microbial composition comprises vitamin Bor a precursor thereof.
 21. The method of claim 1, wherein the microbialcomposition is stable under ambient conditions for at least one week.22. The method of claim 1, wherein the microbial composition is orallyadministered to the ruminant.
 23. The method of claim 22, wherein themicrobial composition is fed to the ruminant.
 24. The method of claim23, wherein the ruminant is fed a step-up diet or a finishing dietcomprising the microbial composition.
 25. The method of claim 1, whereinthe ruminant is a cow or steer.
 26. The method of claim 1, wherein theruminant administered the effective amount of the microbial compositionexhibits a shift in the microbiome of the rumen.
 27. The method of claim1, wherein the one or more desirable traits are selected from the groupconsisting of: a decrease in the incidence and/or prevalence of acidosisor bloat, a decrease in the amount of carbon dioxide and/or carbonicacid in the rumen, an increase in the amount of meat marbling, anincrease in feed efficiency, an increase in feed utilization, anincrease in feed digestibility, a decrease in manure production, anincrease in performance, an increase in weight, an increase inmusculature, a decrease in nitrous oxide production and emission, adecrease in methane production and emission, improved gastrointestinalhealth, and a decrease in mortality.
 28. The method of claim 27, whereinthe one or more desirable traits is an increase in performance.
 29. Themethod of claim 27, wherein the one or more desirable traits is anincrease in feed efficiency.
 30. The method of claim 27, wherein the oneor more desirable traits is a decrease in the incidence and/orprevalence of acidosis or bloat in the ruminant.